Nontypeable Haemophilus Influenzae Research Articles

Non-Typeable Haemophilus influenzae Invade Choroid Plexus Epithelial Cells in a Polar Fashion.

Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood–brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood–cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

Epidemiology and aetiology of community-acquired pneumonia in children: South African Thoracic Society guidelines (part 1)

Background. Pneumonia remains a major cause of morbidity and mortality among South African (SA) children. Improved immunisation regimens, strengthening of HIV programmes, better socioeconomic conditions and new preventive strategies have influenced the epidemiology of pneumonia. Furthermore, sensitive diagnostic tests and better sampling methods in young children improve aetiological diagnosis. Objectives. To summarise current information on childhood community-acquired pneumonia (CAP) epidemiology and aetiology in children as part of the revised South African Thoracic Society guidelines. Methods. The Paediatric Assembly of the South African Thoracic Society and the National Institute for Communicable Diseases expert subgroup on epidemiology and aetiology revised the existing SA guidelines.The subgroup reviewed the published evidence in their area; in the absence of evidence, expert opinion was accepted. Evidence was graded using the British Thoracic Society (BTS) grading system, and the relevant section underwent peer review. Results. Respiratory viruses, particularly respiratory syncytial virus, are the key pathogens associated with hospitalisation for radiologically confirmed pneumonia in HIV-uninfected children. Opportunistic organisms, including Pneumocystis jirovecii , are important pathogens in HIV-infected infants, while non-typable Haemophilus influenzae and Staphylococcus aureus are important in older HIV-infected children. Co-infections with bacteria or other respiratory viruses are common in hospitalised children. Mycobacterium tuberculosis is common in children hospitalised with CAP in SA. Conclusions. Numerous public health measures, including changes in immunisation schedules and expansion of HIV prevention and treatment programmes, have influenced the epidemiology and aetiology of CAP in SA children. These changes have necessitated a revision of the South African Paediatric CAP guidelines, further sections of which will be published as part of a CME series in SAMJ .

Whole-genome analyses reveal gene content differences between nontypeable Haemophilus influenzae isolates from chronic obstructive pulmonary disease compared to other clinical phenotypes.

Nontypeable Haemophilus influenzae (NTHi) colonizes human upper respiratory airways and plays a key role in the course and pathogenesis of acute exacerbations of chronic obstructive pulmonary disease (COPD). Currently, it is not possible to distinguish COPD isolates of NTHi from other clinical isolates of NTHi using conventional genotyping methods. Here, we analysed the core and accessory genome of 568 NTHi isolates, including 40 newly sequenced isolates, to look for genetic distinctions between NTHi isolates from COPD with respect to other illnesses, including otitis media, meningitis and pneumonia. Phylogenies based on polymorphic sites in the core-genome did not show discrimination between NTHi strains collected from different clinical phenotypes. However, pan-genome-wide association studies identified 79 unique NTHi accessory genes that were significantly associated with COPD. Furthermore, many of the COPD-related NTHi genes have known or predicted roles in virulence, transmembrane transport of metal ions and nutrients, cellular respiration and maintenance of redox homeostasis. This indicates that specific genes may be required by NTHi for its survival or virulence in the COPD lung. These results advance our understanding of the pathogenesis of NTHi infection in COPD lungs.

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

Nontypeable Haemophilus influenzae (NTHi) is a chief pathogen in both acute otitis media and otitis media with effusion. Phosphorylcholine (ChoP) is expressed on lipooligosaccharides, and ChoP has phase variation, which is related to its adhesion to and invasion of epithelial cells in the upper airway. However, little is known about the role of ChoP expression. We examined the kinetics of the mucosal clearance of NTHi from the nose and middle ear and the mucosal immune response to NTHi infection by comparing ChoP(+) and ChoP(-) strains in a mouse model of middle ear and nasal challenge. Six-week-old male BALB/c mice were subjected to bacterial challenge in the middle ear and nasopharynx. Mice were inoculated with a suspension of a ChoP(+) strain or ChoP(-) strain of NTHi. On days 1, 3, and 7 after inoculation, the middle ear wash (MEW) and nasal wash (NW) were harvested from each group. The samples were used for bacterial counts and the supernatant was used to measure the level of cytokines and C-reactive protein (CRP). MEWs in the ChoP(+) strain group had significantly higher bacterial counts than those in the ChoP(-) strain group on day 1. However, bacteria were eradicated in the ChoP(+) strain group on day 7. NWs in the ChoP(+) strain group had higher bacterial counts than those in the ChoP(-) strain group during the experiment, however, there was no significant difference between the two strains. The levels of cytokines were significantly higher in the ChoP(-) strain group than in the ChoP(+) strain group in MEWs, but these cytokine levels were low in NWs. The CRP concentration in the ChoP(-) group was high on day 7 in the MEWs. In NWs, the CRP concentration was low in all groups during the experiment. ChoP expression of NTHi changes the organism susceptible to killing by CRP, and the ChoP(+) strain might be gradually eradicated from the middle ear via the CRP-complement cascade, but not from nasopharynx. Based on our findings, phase variation by altering Phosphorylcholine expression of nontypeable Haemophilus influenzae affects bacteria clearance and mucosal immune response in the middle ear and nasopharynx.

Invasive non-typeable Haemophilus influenzae infection due to endometritis associated with adenomyosis

BackgroundThe widespread administration of the Haemophilus influenzae type b vaccine has led to the predominance of non-typable H. influenzae (NTHi). However, the occurrence of invasive NTHi infection based on gynecologic diseases is still rare.Case presentationA 51-year-old Japanese woman with a history of adenomyoma presented with fever. Blood cultures and a vaginal discharge culture were positive with NTHi. With the high uptake in the uterus with 67Ga scintigraphy, she was diagnosed with invasive NTHi infection. In addition to antibiotic administrations, a total hysterectomy was performed. The pathological analysis found microabscess formations in adenomyosis.ConclusionsAlthough NTHi bacteremia consequent to a microabscess in adenomyosis is rare, this case emphasizes the need to consider the uterus as a potential source of infection in patients with underlying gynecological diseases, including an invasive NTHi infection with no known primary focus.

Antimicrobial Photodynamic Therapy with Chlorin e6 Is Bactericidal against Biofilms of the Primary Human Otopathogens.

Moraxella catarrhalis, Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi) are ubiquitous upper respiratory opportunistic pathogens. Together, these three microbes are the most common causative bacterial agents of pediatric otitis media (OM) and have therefore been characterized as the primary human otopathogens. OM is the most prevalent bacterial infection in children and the primary reason for antibiotic administration in this population. Moreover, biofilm formation has been confirmed as a primary mechanism of chronic and recurrent OM disease. As bacterial biofilms are inherently metabolically recalcitrant to most antibiotics and these complex structures also present a significant challenge to the immune system, there is a clear need to identify novel antimicrobial approaches to treat OM infections. In this study, we evaluated the potential efficacy of antibacterial photodynamic therapy (aPDT) with the photosensitizer chlorin e6 (Ce6) against planktonic as well as biofilm-associated M. catarrhalis, S. pneumoniae, and NTHi. Our data indicate aPDT with Ce6 elicits significant bactericidal activity against both planktonic cultures and established biofilms formed by the three major otopathogens (with an efficacy of ≥99.9% loss of viability). Notably, the implementation of a novel, dual-treatment aPDT protocol resulted in this disinfectant effect on biofilm-associated bacteria and, importantly, inhibited bacterial regrowth 24 h posttreatment. Taken together, these data suggest this novel Ce6-aPDT treatment may be a powerful and innovative therapeutic strategy to effectively treat and eradicate bacterial OM infections and, significantly, prevent the development of recurrent disease.IMPORTANCE Otitis media (OM), or middle ear disease, is the most prevalent bacterial infection in children and the primary reason for antibiotic use and surgical intervention in the pediatric population. Biofilm formation by the major bacterial otopathogens, Moraxella catarrhalis, Streptococcus pneumoniae, and nontypeable Haemophilus influenzae, has been shown to occur within the middle ears of OM patients and is a key factor in the development of recurrent disease, which may result in hearing impairment and developmental delays. Bacterial biofilms are inherently impervious to most antibiotics and present a significant challenge to the immune system. In this study, we demonstrate that antimicrobial photodynamic therapy (aPDT) using the photosensitizer chlorin e6 elicits significant bactericidal activity versus planktonic and biofilm-associated otopathogens and supports further analyses of this novel, efficacious, and promising technology as an adjunctive treatment for acute and recurrent OM.

Exposure to welding fumes suppresses the activity of T-helper cells

Welders have an increased susceptibility to airway infections with non-typeable Haemophilus influenzae (NTHi), which implicates immune defects and might promote pneumonia and chronic obstructive pulmonary disease (COPD). We hypothesized that welding-fume exposure suppresses Th1-lymphocyte activity. Non-effector CD4+ T-cells from blood of 45 welders (n = 23 gas metal arc welders, GMAW; n = 16 tungsten inert gas welders, TIG; n = 6 others) and 25 non-welders were ex vivo activated towards Th1 via polyclonal T-cell receptor stimulation and IL-12 (first activation step) and then stimulated with NTHi extract or lipopolysaccharide (LPS) (second activation step). IFNγ and IL-2 were measured by ELISA. In the first activation step, IFNγ was reduced in welders compared to non-welders and in the GMAW welders with higher concentrations of respirable particles compared to the lower exposed TIG welders. IFNγ was not influenced by tobacco smoking and correlated negatively with welding-fume exposure, respirable manganese, and iron. In the second activation step, NTHi and LPS induced additional IFNγ, which was reduced in current smokers compared to never smokers in welders as well as in non-welders. Analyzing both activation steps together, IFNγ production was lowest in smoking welders and highest in never smoking non-welders. IL-2 was not associated with any of these parameters. Welding-fume exposure might suppress Th1-based immune responses due to effects of particulate matter, which mainly consists of iron and manganese. For responses to NTHi this is strongest in smoking welders because welding fume suppresses T-cell activation towards Th1 and cigarette smoke suppresses the subsequent Th1-response to NTHi via LPS. Both effects are independent from IL-2-regulated T-cell proliferation. This might explain the increased susceptibility to infections and might promote COPD development.

Targeting a bacterial DNABII protein with a chimeric peptide immunogen or humanised monoclonal antibody to prevent or treat recalcitrant biofilm-mediated infections.

Background:Chronic and recurrent bacterial diseases are recalcitrant to treatment due to the ability of the causative agents to establish biofilms, thus development of means to prevent or resolve these structures are greatly needed. Our approach targets the DNABII family of bacterial DNA-binding proteins, which serve as critical structural components within the extracellular DNA scaffold of biofilms formed by all bacterial species tested to date. DNABII-directed antibodies rapidly disrupt biofilms and release the resident bacteria which promote their subsequent clearance by either host immune effectors or antibiotics that are now effective at a notably reduced concentration.Methods:First, as a therapeutic approach, we used intact IgG or Fab fragments against a chimeric peptide immunogen designed to target protective epitopes within the DNA-binding tip domains of integration host factor to disrupt established biofilms in vitro and to mediate resolution of existing disease in vivo. Second, we performed preventative active immunisation with the chimeric peptide to induce the formation of antibody that blocks biofilm formation and disease development in a model of viral-bacterial superinfection. Further, toward the path for clinical use, we humanised a monoclonal antibody against the chimeric peptide immunogen, then characterised and validated that it maintained therapeutic efficacy.Findings:We demonstrated efficacy of each approach in two well-established pre-clinical models of otitis media induced by the prevalent respiratory tract pathogen nontypeable Haemophilus influenzae, a common biofilm disease.Interpretation:Collectively, our data revealed two approaches with substantive efficacy and potential for broad application to combat diseases with a biofilm component.FundingSupported by R01 DC011818 to LOB and SDG.

The effect of pneumococcal conjugate vaccines on otitis media from 2005 to 2013 in children aged ≤5 years: a retrospective cohort study in two Swedish regions

ABSTRACT Seven-valent pneumococcal conjugate vaccine (PCV7) was introduced to Sweden in 2009 and replaced by pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) or 13-valent PCV (PCV13) from late 2009. A retrospective cohort study assessed the impact of PCVs on otitis media/acute otitis media (OM) in children aged ≤5 years (NCT02742753) living in Skåne (PCV7 then PHiD-CV) or Västra Götalandsregionen (PCV7 then PCV13) between 2005 and 2013 using linked regional and national databases. Time-series analyses described differences between pre-PCV and post-PCV eras. Adjusted age-period-cohort (APC) predictive models estimated vaccine effectiveness and OM incidence ratios between PCV cohorts. Time-to-first OM diagnosis was estimated in ≤2 year-olds by survival analysis using a Cox proportional hazards model. Descriptive interrupted time-series analyses showed OM incidence in ≤2 year-olds declined by 42% (Skåne) and 25% (Västra Götalandsregionen) after PHiD-CV/PCV13, respectively, versus pre-PCV, but baseline OM incidence and duration of PCV7 use differed between regions. In adjusted APC models, OM incidence decreased after PHiD-CV by 9.9% (95% confidence interval [CI]: 4.4; 15.1, p < .001) and PCV13 by 2.3% (95%CI: −3.2; 7.6, p = .401) compared with pre-PCV. Both PHiD-CV and PCV13 decreased the risk of first OM diagnosis: hazard ratio (95%CI) for PHiD-CV relative to pre-PCV 0.67 (0.65; 0.69); 0.87 (0.85; 0.89) for PCV13 relative to pre-PCV; p < .001 for both comparisons. Within the limitations of this study conducted in two large Swedish regions, descriptive time-series analyses showed that OM incidence rates declined following the introduction of PHiD-CV and PCV13; however, this reduction only reached statistical significance for PHiD-CV in the adjusted APC models.

Detection of Cell-Dissociated Non-Typeable Haemophilus influenzae in the Airways of Patients with Chronic Obstructive Pulmonary Disease.

BackgroundNon-typeable Haemophilus influenzae (NTHi) is the most commonly found pathogen in the lower respiratory airways of patients with COPD. NTHi is predominantly regarded as an intracellular pathogen; however, like most pathogens, it can exist and co-exist in two broad forms: cell-associated (intracellularly or adhered to cells) or cell-dissociated (biofilm dispersed or planktonic). We sought to investigate if cell-dissociated NTHi can be detected from the sputum of COPD patients and assess this relationship to disease severity and airway inflammation.MethodsDNA was extracted from the sputum plug and cell-free supernatant to quantify absolute (cell-associated and cell-dissociated NTHi) and cell-dissociated NTHi, respectively, from 87 COPD subjects attending an observational longitudinal COPD exacerbation study. NTHi was quantified using TaqMan hydrolysis probes, targeting the OMP P6 gene using qPCR.ResultsAt stable state cell-dissociated NTHi was detected 56% of subjects with a median (IQR) of 9.95x102 gene copies (1.26x102 to 1.90x104). Cell-dissociated NTHi correlated with absolute NTHi levels (r=0.34, p<0.01) but not airway inflammation or spirometry at stable state. At exacerbation, cell-dissociated NTHi correlated with lung function (FEV1 r=0.629, p=0.005; FEV1%predicted r=0.564, p=0.015; FVC r=0.476 p=0.046) and sputum neutrophilic inflammation (% neutrophils r=0.688, p=0.002; total neutrophils r=0.518, p=0.028).ConclusionIn patients with COPD, NTHi can exist in both cell-associated and cell-dissociated forms. Cell-dissociated NTHi is associated with neutrophilic airway inflammation during exacerbations of COPD and may be a driving factor in worsening lung function during these episodes.

COPD-Related Modification to the Airway Epithelium Permits Intracellular Residence of Nontypeable Haemophilus influenzae and May Be Potentiated by Macrolide Arrest of Autophagy.

IntroductionCOPD is an inflammatory airway pathology associated with recurrent infection by nontypeable Haemophilus influenzae (NTHi) that is not effectively managed by macrolide antibiotic therapy. We hypothesised that NTHi is able to reside intracellularly within COPD-derived airway epithelial cells (AEC), and that the factors contained in cigarette smoke when coupled with exposure to erythromycin or azithromycin arrest autophagy, the principle mechanism responsible for clearing intracellular bacteria (called “xenophagy”).MethodsCultures of bronchial airway epithelial cells derived from control and COPD participants were differentiated at an air–liquid interface and exposed to macrolide antibiotics, 10% cigarette smoke-extract (CSE) and NTHi. Markers of autophagic flux and intracellular NTHi were assessed using Western blot analysis and transmission electron microscopy.ResultsAEC treated with macrolide antibiotics or 10% CSE exhibited a block in autophagic flux as evidenced by a concomitant increase in LC3-II and Sequestosome abundance (vs control; both P < 0.01). While control AEC showed no clear evidence of intracellular NTHi, COPD-derived cultures exhibited abundant NTHi within the cytoplasm. Further, intracellular NTHi that were encapsulated within vesicles propagated from the apical epithelial layer to the basal cell layer.DiscussionTaken together, our findings indicate that COPD, cigarette smoke and macrolide antibiotics potentiate the susceptibility to persistent intracellular NTHi. A major mechanism for this is arresting normal autophagic flux in airway epithelial cells. Hence, structural modifications that mitigate this off-target effect of macrolides have significant potential to clear intracellular NTHi and thereby reduce the influence of this pathogen in the airways afflicted by COPD.

P187 Unravelling the contribution to the pro-inflammatory burden caused by the persistence of nontypeable haemophilus influenzae in cystic fibrosis

P187 Unravelling the contribution to the pro-inflammatory burden caused by the persistence of nontypeable haemophilus influenzae in cystic fibrosis

Nontypeable Haemophilus influenzae Responds to Virus-Infected Cells with a Significant Increase in Type IV Pilus Expression.

Nontypeable Haemophilus influenzae (NTHI) colonizes the human nasopharynx, but when the host immune response is dysregulated by upper respiratory tract (URT) virus infection, NTHI can gain access to more distal airway sites and cause disease. The NTHI type IV pilus (T4P) facilitates adherence, benign colonization, and infection, and its majority subunit PilA is in clinical trials as a vaccinogen. To further validate the strategy of immunization with PilA against multiple NTHI-induced diseases, it is important to demonstrate T4P expression under microenvironmental conditions that predispose to NTHI infection of the airway. Because URT infection commonly facilitates NTHI-induced diseases, we examined the influence of ongoing virus infection of respiratory tract epithelial cells on NTHI T4P expression in vitro Polarized primary human airway epithelial cells (HAEs) were sequentially inoculated with one of three common URT viruses, followed by NTHI. Use of a reporter construct revealed that NTHI upregulated pilA promoter activity when cultured with HAEs infected with adenovirus (AV), respiratory syncytial virus (RSV), or rhinovirus (RV) versus that in mock-infected HAEs. Consistent with these results, pilA expression and relative PilA/pilin abundance, as assessed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblot, respectively, were also significantly increased when NTHI was cultured with virus-infected HAEs. Collectively, our data strongly suggest that under conditions of URT virus infection, PilA vaccinogen induction of T4P-directed antibodies is likely to be highly effective against multiple NTHI-induced diseases by interfering with T4P-mediated adherence. We hypothesize that this outcome could thereby limit or prevent the increased load of NTHI in the nasopharynx that characteristically precedes these coinfections.IMPORTANCE Nontypeable Haemophilus influenzae (NTHI) is the predominant bacterial causative agent of many chronic and recurrent diseases of the upper and lower respiratory tracts. NTHI-induced chronic rhinosinusitis, otitis media, and exacerbations of cystic fibrosis and chronic obstructive pulmonary disease often develop during or just after an upper respiratory tract viral infection. We have developed a vaccine candidate immunogen for NTHI-induced diseases that targets the majority subunit (PilA) of the type IV twitching pilus (T4P), which NTHI uses to adhere to respiratory tract epithelial cells and that also plays a role in disease. Here, we showed that NTHI cocultured with virus-infected respiratory tract epithelial cells express significantly more of the vaccine-targeted T4P than NTHI that encounters mock-infected (healthy) cells. These results strongly suggest that a vaccine strategy that targets the NTHI T4P will be effective under the most common predisposing condition: when the human host has a respiratory tract virus infection.

MUC5B induces in vitro neutrophil extracellular trap formation: Implication in otitis media.

BackgroundChronic otitis media (COM) is characterized by middle ear fluid predominantly containing cytokines, Nontypeable haemophilus influenzae (NTHi), the mucin MUC5B, and neutrophil extracellular traps (NETs). NETs consist of extracellular DNA coated with antibacterial proteins such as myeloperoxidase (MPO) and citrullinated histone 3 (CitH3). NETs can damage tissues and sustain inflammation. Our study aimed to develop an in vitro model of NETosis, testing COM inductors.MethodsNETosis was evaluated in fresh blood human neutrophils attached to collagen‐coated plates and in suspension exposed to phorbol myristate acetate (PMA) as a control, and COM relevant mediators. Confocal microscopy, DNA fluorescence assay and flow cytometry were used to quantify NETosis.ResultsPMA exposure induced DNA, MPO, and CitH3 by immunofluorescence (IF) most significantly at 3 hours (3.8‐fold for DAPI, 7.6‐fold for MPO, and 6.9‐fold for CitH3, all P < .05). IL‐8 and TNF‐α cytokines showed milder increases of DAPI, MPO, and CitH3 positive cells. NTHi had no effect on these NETs markers. Purified salivary MUC5B (10 to 40 μg/mL) produced potent increases, comparable to PMA. A composite NET score summing the fold‐increases for DAPI, MPO, and CitH3 demonstrated PMA at 13.6 to 19 relative to control set at 1; and MUC5B at 8.6 to 16.3 (all P < .05). IL‐8 and TNF‐α showed scores of 5.4 and 3, respectively, but these were not statistically significant.ConclusionWe developed a reliable in vitro assay for NETosis which demonstrated that salivary MUC5B is a potent inductor of NETs whereas IL‐8, TNF‐α, live and lyzed NTHi demonstrated minimal to no NETosis.Level of evidenceNA.

The Jeff Mouse Mutant Model for Chronic Otitis Media Manifests Gain-of-Function as Well as Loss-of-Function Effects.

Chronic otitis media (OM) is the most common cause of hearing loss worldwide, yet the underlying genetics and molecular pathology are poorly understood. The mouse mutant Jeff is a single gene mouse model for OM identified from a deafness screen as part of an ENU mutagenesis program at MRC Harwell. Jeff carries a missense mutation in the Fbxo11 gene. Jeff heterozygotes (Fbxo11Jf/+) develop chronic OM at weaning and have reduced hearing. Homozygotes (Fbxo11Jf/Jf) display perinatal lethality due to developmental epithelial abnormalities. In order to investigate the role of FBXO11 and the type of mutation responsible for the phenotype of the Jeff mice, a knock-out mouse model was created and compared to Jeff. Surprisingly, the heterozygote knock-outs (Fbxo11tm2b/+) show a much milder phenotype: they do not display any auditory deficit and only some of them have thickened middle ear epithelial lining with no fluid in the ear. In addition, the knock-out homozygote embryos (Fbxo11tm2b/tm2b), as well as the compound heterozygotes (Fbxo11tm2b/Jf) show only mild abnormalities compared to Jeff homozygotes (Fbxo11Jf/Jf). Interestingly, 3 days after intranasal inoculation of the Fbxo11tm2b/+ mice with non-typeable Haemophilus influenzae (NTHi) a proportion of them have inflamed middle ear mucosa and fluid accumulation in the ear suggesting that the Fbxo11 knock-out mice are predisposed to NTHi induced middle ear inflammation. In conclusion, the finding that the phenotype of the Jeff mutant is much more severe than the knock-out indicates that the mutation in Jeff manifests gain-of-function as well as loss-of-function effects at both embryonic and adult stages.

Draft Genome Sequence of an Isolate of Nontypeable Haemophilus influenzae from an Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Tasmania.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of human illness, including pneumonia and acute exacerbations of chronic obstructive pulmonary disease (COPD). We report here the draft genome of an isolate of NTHi collected from the sputum of a patient presenting with COPD in Tasmania, Australia.

10-Valent pneumococcal non-typeable H. influenzae protein D conjugate vaccine (PHiD-CV10) versus 13-valent pneumococcal conjugate vaccine (PCV13) as a booster dose to broaden and strengthen protection from otitis media (PREVIX_BOOST) in Australian Aboriginal children: study protocol for a randomised controlled trial

IntroductionStreptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) are major otitis media pathogens that densely co-colonise the nasopharynx and infect the middle ear of Australian Aboriginal infants from very early in...

A longitudinal hospital-based epidemiology study to assess acute otitis media incidence and nasopharyngeal carriage in Korean children up to 24 months

ABSTRACT This study was conducted to assess the nasopharyngeal (NP) carriage and acute otitis media (AOM) occurrence in Korean children who received pneumococcal conjugate vaccines (PCVs). The longitudinal study was conducted through four consecutive visits. At each visit, NP aspirates were obtained and subjects were asked to visit if AOM occurred. A total of 305 subjects were enrolled and received PCV13 (n = 182) or PCV10 (n = 123). In the PCV13 group, the NP carriage of Streptococcus pneumoniae at each visit was 2.7%, 14.8%, 18.7%, and 15.9%, respectively. Non-typeable Haemophilus influenzae (NTHi) was 3.3%, 2.7%, 2.7%, and 5.5%, and that of Moraxella catarrhalis was 1.1%, 9.3%, 4.9%, and 0.5%. In the PCV10 group, the NP carriage of S. pneumoniae at each visit was 3.3%, 7.3%, 6.5%, and 4.1%, respectively. That of NTHi was 2.4%, 4.1%, 1.6%, and 0.8%, and that of M. catarrhalis was 4.1%, 0.8%, 0.8%, and 0.0%. AOM occurrence in the PCV13 group observed after the primary dose and before booster dose was 20.9%, occurrence after booster dose was 11.0%, and the incidence of two or more AOM was 11.0%. In the PCV10 group, AOM occurrence was 9.8%, 7.3%, respectively, and the incidence of two or more AOM was 2.4%. The predominant S. pneumoniae isolated were non-vaccine type (10A, 15A, and 15B). In this study, AOM occurrence was lower in the PCV10 group than in the PCV13 group. This seems to be related to ecological changes that lead to differences in NP carriage, especially S. pneumoniae and NTHi.

Effects of Amniotic Membrane Extract on the Hyperplastic Response of the Middle Ear Mucosa in a Bacterially-Induced Otitis Media Rat Model: A Preliminary Study.

ObjectivesHuman amniotic membrane extract (AME) is known to contain numerous bioactive factors and anti-inflammatory substances. However, the anti-inflammatory effects of AME on the middle ear (ME) mucosa are unclear. This study assessed the effects of AME on the growth of the ME mucosa in response to bacterially-induced otitis media (OM).MethodsOM was induced by inoculating nontypeable Haemophilus influenzae (NTHi) into the ME cavity of rats. ME mucosal explants were cultured in AME concentrations of 0, 5, 10, or 50 μg/mL. The area of explant outgrowth was measured in culture and analyzed at 1, 3, 5, and 7 days after explantation. The expression of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the explants was also evaluated using quantitative polymerase chain reaction (PCR) and immunocytochemistry (ICC).ResultsThe NTHi-induced ME mucosa growth increased gradually over the 7-day culture period in all explants at different AME concentrations. There was a trend for mucosal growth inhibition at higher concentrations of AME, although the growth was not significantly different among the groups until day 5. The ME mucosal explants treated with the 50 μg/mL concentration of AME showed significantly suppressed growth on postexplantation day 7 compared with other explants on the same day. PCR and ICC staining revealed that the expression of Ki-67, MUC5AC, TNF-α, and IL-10 further decreased in the explants with higher concentrations of AME than in those with lower concentrations of AME.ConclusionOur results showed that higher concentrations of AME reduced the mucosal proliferative response in bacterial OM in rats. These findings provide evidence that AME has an influence on the inflammatory and proliferative responses to NTHi infection in ME mucosa.

Elucidating the Protein‐Protein Interactions between Nontypeable Haemophilus Influenzae Outer Membrane Proteins Omp26 and Protein D

Two leading protein vaccine candidates from Nontypeable Haemophilus Influenzae (NTHi) are outer membrane proteins Omp26 and Protein D. When administered individually, Omp26 and Protein D elicit an antibody response in mice; when combined, the Protein D antibody response is significantly suppressed. Here, we describe a biochemical approach toward elucidating the interactions between Omp26 and Protein D, which we propose contribute to the suppression of Protein D‐specific antibody production. Preliminary data from enzyme‐linked immunosorbent assays and other standard protein detection methods suggest that Protein D binds strongly to Omp26 in vitro and perhaps in vivo in mice.