Nontypeable Haemophilus Influenzae Research Articles

Comparative Genomic Study of Non-typeable Haemophilus influenzae in Children with Pneumonia and Healthy Controls

Comparative Genomic Study of Non-typeable Haemophilus influenzae in Children with Pneumonia and Healthy Controls

Invasive Nontypeable Haemophilus influenzae Disease Outbreak at an Elementary School—Michigan, May 2023

Invasive Nontypeable Haemophilus influenzae Disease Outbreak at an Elementary School—Michigan, May 2023

Haemophilus influenzae and pneumococci: Co-colonization, interactions, cooperation and competition.

Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae (pneumococcus) are pathobionts that share common environmental niches within the upper respiratory tract. They can form part of the resident upper airway microbiota, but under certain environmental circumstances become pathogenic and induce disease. In children, both organisms have a considerable impact on the healthcare system, commonly causing acute otitis media and pneumonia. They are also associated with chronic biofilm-mediated respiratory infections, such as persistent middle ear effusions and chronic suppurative otitis media, and in the lower airways with protracted bacterial bronchitis and bronchiectasis. Consequently, both organisms are responsible for large numbers of antibiotic prescriptions and substantial healthcare costs. The complex relationship between NTHi and pneumococcal co-interaction during colonization, infection and biofilm formation is poorly understood and a greater understanding is needed to facilitate development of future therapies, and novel interventions and prevention strategies. Co-infections with both bacteria can result in more severe disease, with disease severity likely mediated by their ability to cooperate in some in vivo niches. However, this relationship is not always straightforward, as under certain conditions, these two bacteria compete rather than cooperate. Current opinion supports developing a vaccine targeting NTHi strains, as well as a combined vaccine targeting both NTHi and pneumococci to decrease the respiratory disease burden in young children. This review summarizes our current knowledge of the interactions between NTHi and pneumococci and speculates on the future directions of research to understand how these bacteria co-exist and how to better prevent and treat NTHi and pneumococcal infection.

The emergence of transposon-driven multidrug resistance in invasive nontypeable Haemophilus influenzae over the last decade

Nontypeable Haemophilus influenzae (NTHi), once considered a harmless commensal, has emerged as a significant concern due to the increased prevalence of multidrug-resistant (MDR) strains and their association with invasive infections. This study aimed to explore the epidemiology and molecular resistance mechanisms of 51 NTHi isolates collected from patients with invasive infections in northern Taiwan between 2011 and 2020. This investigation revealed substantial genetic diversity, encompassing 29 distinct sequence types and 18 clonal complexes. Notably, 68.6% of the isolates exhibited ampicillin resistance, with 28 categorised as MDR and four isolates were even resistant to up to six antibiotic classes. Among the MDR isolates, 18 pulsotypes were identified, indicating diverse genetic lineages. Elucidation of their resistance mechanisms revealed 18 β-lactamase-producing amoxicillin–clavulanate-resistant (BLPACR) isolates, 12 β-lactamase-producing ampicillin-resistant (BLPAR) isolates, and 5 β-lactamase-nonproducing ampicillin-resistant (BLNAR) isolates. PBP3 analysis revealed 22 unique substitutions in BLPACR and BLNAR, potentially contributing to cephem resistance. Notably, novel transposons, Tn7736-Tn7739, which contain critical resistance genes, were discovered. Three strains harboured Tn7739, containing seven resistance genes [aph(3′)-Ia, blaTEM-1, catA, sul2, strA, strB, and tet(B)], while four other strains carried Tn7736, Tn7737, and Tn7738, each containing three resistance genes [blaTEM-1, catA, and tet(B)]. The emergence of these novel transposons underscores the alarming threat posed by highly resistant NTHi strains. Our findings indicated that robust surveillance and comprehensive genomic studies are needed to address this growing public health challenge.

Invasive Nontypeable Haemophilus influenzae Disease Outbreak at an Elementary School - Michigan, May 2023.

In May 2023, the Detroit Health Department was notified of four cases of invasive nontypeable Haemophilus influenzae (Hi) disease among students attending the same elementary school and grade, all with illness onsets within 7 days. Three patients were hospitalized, and one died. Most U.S. cases of invasive Hi disease are caused by nontypeable strains. No vaccines against nontypeable or non-type b Hi strains are currently available. Chemoprophylaxis is not typically recommended in response to nontypeable Hi cases; however, because of the high attack rate (four cases among 46 students; 8.7%), rifampin prophylaxis was recommended for household contacts of patients with confirmed cases and for all students and staff members in the school wing where confirmed cases occurred. Only 10.8% of students for whom chemoprophylaxis was recommended took it, highlighting gaps in understanding among caregivers and health care providers about persons for whom chemoprophylaxis was recommended. Public health authorities subsequently enhanced communication and education to the school community, improved coordination with health care partners, and established mass prophylaxis clinics at the school. This outbreak highlights the potential for nontypeable Hi to cause serious illness and outbreaks and the need for chemoprophylaxis guidance for nontypeable Hi disease. Achieving high chemoprophylaxis coverage requires education, communication, and coordination with community and health care partners.

Biofilm Production and Its Implications in Pediatrics.

Biofilms, aggregates of bacteria enclosed in a self-produced matrix, have been implicated in various pediatric respiratory infections, including acute otitis media (AOM), otitis media with effusion (OME), adenoiditis, protracted bacterial bronchitis, and pulmonary exacerbations in cystic fibrosis. These infections are prevalent in children and often associated with biofilm-producing pathogens, leading to recurrent and chronic conditions. Biofilms reduce antibiotic efficacy, contributing to treatment failure and disease persistence. This narrative review discusses biofilm production by respiratory pathogens such as Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Pseudomonas aeruginosa, and Staphylococcus aureus. It examines their mechanisms of biofilm formation, antibiotic resistance, and the challenges they present in clinical treatment. Various antibiofilm strategies have shown promise in vitro and in animal studies, including the use of N-acetylcysteine, enzymes like dispersin B, and agents disrupting quorum sensing and biofilm matrix components. However, their clinical application, particularly in children, remains limited. Traditional treatments for biofilm-associated diseases have not significantly evolved, even with biofilm detection. The transition from experimental findings to clinical practice is complex and requires robust clinical trials and standardized biofilm detection protocols. Addressing biofilms in pediatric respiratory infections is crucial for improving treatment outcomes and managing recurrent and chronic diseases effectively.

A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Nontypeable Haemophilus influenzae challenge during gammaherpesvirus infection enhances viral reactivation and latency

Gammaherpesviruses are ubiquitous, lifelong pathogens associated with multiple cancers that infect over 95% of the adult population. Increases in viral reactivation, due to stress and other unknown factors impacting the immune response, frequently precedes lymphomagenesis. One potential stressor that could promote viral reactivation and increase viral latency would be the myriad of infections from bacterial and viral pathogens that we experience throughout our lives. Using murine gammaherpesvirus 68 (MHV68), a mouse model of gammaherpesvirus infection, we examined the impact of bacterial challenge on gammaherpesvirus infection. We challenged MHV68 infected mice during the establishment of latency with nontypeable Haemophilus influenzae (NTHi) to determine the impact of bacterial infection on viral reactivation and latency. Mice infected with MHV68 and then challenged with NTHi, saw increases in viral reactivation and viral latency. These data support the hypothesis that bacterial challenge can promote gammaherpesvirus reactivation and latency establishment, with possible consequences for viral lymphomagenesis.

An antibacterial, multifunctional nanogel for efficient treatment of neutrophilic asthma

Asthma is a chronic and heterogeneous disease affecting the lungs and respiratory tract. In particular, the neutrophil subtype of asthma was described as persistent, more severe, and corticosteroid-resistant. Growing evidence suggested that nontypeable Haemophilus influenzae (NTHi) infection contributes to the development of neutrophilic asthma, exacerbating clinical symptoms and increasing the associated medical burden. In this work, arginine-grafted chitosan (CS-Arg) was ionically cross-linked with tris(2-carboxyethyl) phosphine (TCEP), and a highly-efficient antimicrobial agent, poly-ε-L-Lysine (ε-PLL), was incorporated to prepare ε-PLL/CS-Arg/TCEP (ECAT) composite nanogels. The results showed that ECAT nanogels exhibited highly effective inhibition against the proliferation of NTHi, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In addition, ECAT nanogels could effectively inhibit the formation of mucins aggregates in vitro, suggesting that the nanogel might have the potential to destroy mucin in respiratory disease. Furthermore, in the ovalbumin (OVA)/NTHi-induced Balb/c mice model of neutrophilic asthma, the number of neutrophils in the alveolar lavage fluid and the percentage of inflammatory cells in the blood were effectively reduced by exposure to tower nebulized administration of ECAT nanogels, and reversing airway hyperresponsiveness (AHR) and reducing inflammation in neutrophilic asthma mice. In conclusion, the construction of ECAT nanogels was a feasible anti-infective and anti-inflammatory therapeutic strategy, which demonstrated strong potential in the clinical treatment of neutrophilic asthma.

Orbital complications of pediatric acute rhinosinusitis in the pneumococcal conjugate vaccine era

ObjectivesTo evaluate the impact of the pneumococcal conjugate vaccines (PCVs) introduction on the orbital complications of acute rhino-sinusitis (OC-ARS). MethodsA retrospective cohort study of all pediatric patients with OC-ARS during the period 2002–2019. Data included clinical, demographic, laboratory, and microbiology findings. Patients were divided into three groups: before PCV7 introduction (group 1), after PCV7 and before PCV13 (group 2), and after PCV13 (group 3). ResultsOf 265 enrolled patients, 117, 39, and 109 were assigned to groups 1, 2, and 3. During the study period, a significant decrease was recorded in the percentages of patients in Chandler classification severity category 1, with an increase in patients in category 3 (P = 0.011). The yearly incidence of OC-ARS decreased from 12.64 cases per 100,000 population in 2002 to 5.56 per 100,000 in 2008, and 2.99 per 100,000 in 2019 (P < 0.001). Patients aged 0–4 years showed a dramatic decrease from 29 cases per 100,000 population in 2002 to 4.27 per 100,000 in 2019 (P < 0.001). The pathogens retrieved from all cultures performed were Streptococcus pneumoniae (32.5%), non-typeable Haemophilus influenzae (27.5%), Streptococcus Species, (12.5%), and Staphylococcus aureus (20%), with no changes in distribution during the study periods. Surgery was performed in 28 (10.6%) patients. ConclusionsA significant decrease was seen in the overall incidence of OC-ARS, mainly attributable to the decrease in patients aged 0–4 years. An increase was recorded in the severity of the disease following PCVs introduction.

The A2ml1-Knockout mouse as an animal model for non-syndromic otitis media

BackgroundInflammation and infection of the middle ear, known as otitis media (OM), is a leading cause of hearing loss and the most frequently diagnosed disease in children worldwide. Traditionally, mouse models for OM rely on inducing acute infection through inoculation of the middle ear, e.g. with the human otopathogen non-typeable Haemophilus influenzae (NTHi), and with very few genetic models with spontaneous or chronic OM. A2ML1 variants, including loss-of-function variants, were associated with susceptibility to OM in humans, but no animal model has been reported for A2ml1-related OM. Here, we report our middle ear findings in a mouse line with a CRISPR-induced knockout (KO) of A2ml1. MethodsMice were X-rayed prior to harvest to determine if there are craniofacial or skeletal abnormalities. Tissue from mouse middle ears, as well as other upper respiratory mucosal tissues, were harvested. The harvested middle ear bullae were examined under microscope and submitted for histologic preparation to study phenotypic indications of OM. RNA samples isolated from middle ear tissue were assayed for expression of genes correlated with A2ML1 expression in humans. ResultsData from a total of 119 mice (35 wildtype, 40 heterozygous, 44 homozygous) are presented here, with each analyses being performed on subsets of these mice. There were no significant craniofacial differences by genotype (n = 22). Findings in mice with the A2ml1-KO indicated an increased incidence of OM (n=29; odds ratio = 11; CI: 1.1, 573.6; Fisher exact two-sided p = 0.02) with tympanic membrane perforations or thickening, as well as cases of middle ear effusion, inflammatory cells, or fluid from histologic sections. Dsp was upregulated in the middle ear tissues of homozygous mice (Wilcoxon test p = 0.001). ConclusionThus far, our results in this A2ml1-KO mouse line indicate spontaneous occurrence of OM and dysregulation of Dsp in the middle ear as a potential disease mechanism for A2ml1-related OM.

Modeling airway persistent infection of Moraxella catarrhalis and nontypeable Haemophilus influenzae by using human in vitro models.

Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation, the precise means through which they contribute to disease severity and chronicity remains incompletely understood, posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay in vitro even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work, by using air-liquid-interface (ALI) human airway in vitro models, we aimed to recreate COPD-related persistent bacterial infections. In particular, we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression, allowing one to monitor host-pathogen interactions for up to three weeks. Notably, the use of these models, coupled with confocal and transmission electron microscopy, revealed unique features associated with NTHi and Mcat infection, highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall, this study demonstrates the possibility to perform long term host-pathogen investigations in vitro with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets.

Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded. Clinical Trial Registration: NCT02075541

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.

A Novel Catalase Nanogels for Effective Treatment of Neutrophilic Asthma

AbstractNeutrophilic asthma, as corticosteroid resistant asthma, is clinically more severe than eosinophilic asthma. Therefore, the treatment of neutrophilic asthma has been a challenging. Reactive oxygen species (ROS) play a key role in the neutrophilic asthma. However, the direct use of antioxidant enzymes, such as catalase (CAT), is challenging due to the poor stability, short plasma half‐life, and the lack of effective drug delivery systems to pulmonary systems. In this work, a novel kind of nanogels for delivering CAT (CAT‐NGs) is synthesized, incorporating CAT with an antibacterial “protective film”, as a modality for the treatment of neutrophilic asthma. Compared with free CAT, CAT‐NGs demonstrated superior enzyme activity and trypsin resistance, and exhibited outstanding anti‐inflammatory and antioxidant activities in vitro. Furthermore, CAT‐NGs showed significant inhibitory effects on nontypeable Haemophilus influenzae (NTHi), Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Additionally, nebulized inhalation of CAT‐NGs alleviated pulmonary inflammation in neutrophilic asthma mice. Mechanistically, the alleviation of neutrophilic asthma symptoms by CAT‐NGs is possibly associated with the clearance of ROS in the lung and the inhibition of the NLRP3 and NF‐κB pathways. Hence, the work have demonstrated the therapeutic potential of CAT‐NGs, offering new insights into the clinical treatment of neutrophilic asthma.

Molecular characteristics and biofilm formation capacity of nontypeable Haemophilus influenza strains isolated from lower respiratory tract in children

With the widespread introduction of the Hib conjugate vaccine, Nontypeable Haemophilus influenzae (NTHi) has emerged as the predominant strain globally. NTHi presents a significant challenge as a causative agent of chronic clinical infections due to its high rates of drug resistance and biofilm formation. While current research on NTHi biofilms in children has primarily focused on upper respiratory diseases, investigations into lower respiratory sources remain limited. In this study, we collected 54 clinical strains of lower respiratory tract origin from children. Molecular information and drug resistance features were obtained through whole gene sequencing and the disk diffusion method, respectively. Additionally, an in vitro biofilm model was established. All clinical strains were identified as NTHi and demonstrated the ability to form biofilms in vitro. Based on scanning electron microscopy and crystal violet staining, the strains were categorized into weak and strong biofilm-forming groups. We explored the correlation between biofilm formation ability and drug resistance patterns, as well as clinical characteristics. Stronger biofilm formation was associated with a longer cough duration and a higher proportion of abnormal lung imaging findings. Frequent intake of β-lactam antibiotics might be associated with strong biofilm formation. While a complementary relationship between biofilm-forming capacity and drug resistance may exist, further comprehensive studies are warranted. This study confirms the in vitro biofilm formation of clinical NTHi strains and establishes correlations with clinical characteristics, offering valuable insights for combating NTHi infections.

Nasal Delivery of Haemophilus haemolyticus Is Safe, Reduces Influenza Severity, and Prevents Development of Otitis Media in Mice.

Despite vaccination, influenza and otitis media (OM) remain leading causes of illness. We previously found that the human respiratory commensal Haemophilus haemolyticus prevents bacterial infection in vitro and that the related murine commensal Muribacter muris delays OM development in mice. The observation that M muris pretreatment reduced lung influenza titer and inflammation suggests that these bacteria could be exploited for protection against influenza/OM. Safety and efficacy of intranasal H haemolyticus at 5 × 107 colony-forming units (CFU) was tested in female BALB/cARC mice using an influenza model and influenza-driven nontypeable Haemophilus influenzae (NTHi) OM model. Weight, symptoms, viral/bacterial levels, and immune responses were measured. Intranasal delivery of H haemolyticus was safe and reduced severity of influenza, with quicker recovery, reduced inflammation, and lower lung influenza virus titers (up to 8-fold decrease vs placebo; P ≤ .01). Haemophilus haemolyticus reduced NTHi colonization density (day 5 median NTHi CFU/mL = 1.79 × 103 in treatment group vs 4.04 × 104 in placebo, P = .041; day 7 median NTHi CFU/mL = 28.18 vs 1.03 × 104; P = .028) and prevented OM (17% OM in treatment group, 83% in placebo group; P = .015). Haemophilus haemolyticus has potential as a live biotherapeutic for prevention or early treatment of influenza and influenza-driven NTHi OM. Additional studies will deem whether these findings translate to humans and other respiratory infections.

Preparation and immunogenicity evaluation of C-HapS-P6 fusion protein vaccine against nontypeable Haemophilus influenzae in mice

Nontypeable Haemophilus influenzae (NTHi) is the dominant pathogen in several infectious diseases. Currently the use of antibiotics is the main intervention to prevent NTHi infections, however with the emergence of drug resistant strains, it has compromised the treatment of respiratory infections with antibiotics. Therefore there is an urgent need to develop a safe and effective vaccine to prevent NTHi infections. We investigate the potential of C-HapS-P6 fusion protein as a vaccine for treating NTHi in murine models. PGEX-6P2/C-HapS-P6 fusion gene was constructed using overlap extension polymerase chain reaction. The recombined plasmid was transformed into Escherichia coli for protein expression. The mice were subjected to intraperitoneal immunization using purified antigens. Immunoglobulin (Ig) G in serum samples and IgA in nasal and lung lavage fluids were analyzed using enzyme-linked immunosorbent assay. Cytokine release and proliferation capacity of splenic lymphocytes in response to antigens were measured in vitro. The protective effect of the C-HapS-P6 protein against NTHi infection was evaluated by NTHi count and histological examination. The data showed that the C-HapS-P6 fusion protein increased significantly the levels of serum IgG and nasal and lung IgA, and promoted the release of interleukin (IL)-2, interferon-ϒ, IL-4, IL-5, and IL-17 and the proliferation of splenic lymphocytes compared with C-HapS or P6 protein treatment alone. Moreover, C-HapS-P6 effectively reduced the NTHi colonization in the nasopharynx and lungs of mice. In conclusion, our results demonstrated that the C-HapS-P6 fusion protein vaccine can significantly enhance humoral and cell immune responses and effectively prevent against NTHi infection in the respiratory tract in murine models.

17β-estradiol Attenuates the Middle Ear Inflammatory Response to Nontypeable Haemophilus influenzae.

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

Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.