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Clarithromycin is a 14-member lactone ring macrolide with potent activity against Haemophilus influenzae, including ampicillin-resistant strains. We evaluated the in vivo efficacy of clarithromycin at 40 mg/day and 100 mg/day for 3 days in the treatment of a murine model of pneumonia using a macrolide-resistant H. influenzae strain, which was also ampicillin resistant. The MIC of clarithromycin was 64 microg/ml. The viable bacterial counts in infected tissues after treatment with 100 mg clarithromycin/kg of body weight were lower than the counts obtained in control and 40-mg/kg clarithromycin-treated mice. The concentrations of macrophage inflammatory protein 2 (MIP-2) and interleukin 1beta (IL-1beta) in bronchoalveolar lavage fluid (BALF) samples from mice treated at both concentrations were lower than in the control group. Pathologically, following infection, clarithromycin-treated mice, particularly at a dose of 100 mg/kg, showed lower numbers of neutrophils in alveolar walls, and inflammatory changes had apparently improved, whereas large aggregates of inflammatory cells were observed within the alveoli of control mice. In addition, we demonstrated that clarithromycin has bacteriological effects against intracellular bacteria at levels below the MIC. Our results indicate that clarithromycin may be useful in vivo for macrolide-resistant H. influenzae, and this phenomenon may be related to the good penetration of clarithromycin into bronchoepithelial cells. We also believe that conventional drug susceptibility tests may not reflect the in vivo effects of clarithromycin.

Secondary bacterial infections that follow infection with influenza virus result in considerable morbidity and mortality in young children, the elderly, and immunocompromised individuals and may also significantly increase mortality in normal healthy adults during influenza pandemics. We herein describe a mouse model for investigating the interaction between influenza virus and the bacterium Haemophilus influenzae. Sequential infection with sublethal doses of influenza and H. influenzae resulted in synergy between the two pathogens and caused mortality in immunocompetent adult wild-type mice. Lethality was dependent on the interval between administration of the bacteria and virus, and bacterial growth was prolonged in the lungs of dual-infected mice, although influenza virus titers were unaffected. Dual infection induced severe damage to the airway epithelium and confluent pneumonia, similar to that observed in victims of the 1918 global influenza pandemic. Increased bronchial epithelial cell death was observed as early as 1 day after bacterial inoculation in the dual-infected mice. Studies using knockout mice indicated that lethality occurs via a mechanism that is not dependent on Fas, CCR2, CXCR3, interleukin-6, tumor necrosis factor, or Toll-like receptor-4 and does not require T or B cells. This model suggests that infection with virulent strains of influenza may predispose even immunocompetent individuals to severe illness on secondary infection with H. influenzae by a mechanism that involves innate immunity, but does not require tumor necrosis factor, interleukin-6, or signaling via Toll-like receptor-4.

Nontypeable Haemophilus influenzae (NTHi) is the etiological agent most frequently associated with bacterial exacerbations of chronic obstructive pulmonary disease (COPD). The present work shows that NTHi strains induced in primary normal human bronchial epithelial cells (NHBE) a cytokine/chemokine response in which CCL-5 and CXCL-10 were predominant. Production of both cytokines was inhibited by an anti-TLR3 monoclonal antibody (mAb) in a dose-dependent manner, but not by control human IgG4 antibodies, thus suggesting a TLR3-dependency of the NTHi stimulation. BEAS-2B, an immortalized human bronchial epithelial cell line, also showed a similar NTHi-induced response that was inhibited by the anti-TLR3 mAb. A BEAS-2B cell line stably expressing TLR3 siRNA showed significantly reduced cytokine/chemokine responses to NTHi stimulation, confirming the role of TLR3 in the response. These results indicate that TLR3 is a key component in the response of human bronchial epithelial cells to NTHi, and suggest that cognate neutralizing mAbs might be a useful therapeutic tool to regulate the inflammatory response.

Haemophilus influenzae is a common pathogen of respiratory infections. We examined whether beta-lactamase-negative ampicillin-resistant (BLNAR) strains that are known to have ampicillin resistance due to a substitution of amino acid of penicillin binding protein (PBP)-3, differ from beta-lactamase-negative ampicillin-susceptible strains with regard to invasion of bronchial epithelium. After 3h incubation of each of 34 beta-lactamase-negative ampicillin-susceptible and 57 BLNAR strains in the presence of BEAS-2B cells, a human bronchial epithelium cell line, extracellular bacteria were killed using gentamicin and intracellular bacteria numbered. All nine strains in which the efficiency of invasion was 1% or higher were BLNAR strains. The rate of invasion was significantly greater in strains with PBP-3 amino acid substitution (Met377 to Ile, Ser385 to Thr, Leu389 to Phe, and Asn526 to Lys) (n=34) than in those with no amino acid substitution. Electron microscopy showed that high invasive BLNAR strains were observed in cytoplasm of BEAS-2B cell layer. The injured cells were 9.44+/-1.76% among attaching cells examined by trypan blue staining after 6h. These data may suggest that the amino acid substitution of the PBP in BLNAR strains may at least partly play roles in macropinocytosis, leading to the invasion and injury to epithelial cells.

Background: This study describes the epidemiologic, microbiologic, and otologic features and selected signs and symptoms of acute otitis media (AOM) caused by Moraxella catarrhalis and compares them with AOM caused by other bacterial pathogens., Methods: Patients aged <5 years with culture-positive AOM from whom a middle ear fluid specimen was obtained and cultured during 1999-2006 were enrolled in the study., Results: Of a total of 12,799 AOM episodes, 8198 (64%) were culture positive, with isolation of 10,382 pathogens: Haemophilus influenzae, 4982 (48.0%); Streptococcus pneumoniae, 4450 (42.9%); M. catarrhalis, 501 (4.8%); and group A streptococci, 449 (4.3%). The distribution of single versus mixed M. catarrhalis infection was significantly different compared with the 3 other pathogens (165 cases [32.9%] as a single pathogen of all M. catarrhalis AOM episodes vs 3108 [62.4%] in AOM caused by H. influenzae, 2592 [58.2%] in AOM caused by S. pneumoniae, and 304 [67.7%] in AOM caused by group A streptococci; P < .001 for all comparisons). In multivariate analysis, M. catarrhalis AOM was more frequent in patients experiencing their first AOM episode versus recurrent AOM and mixed infections. M. catarrhalis AOM was associated with lower proportions of spontaneous perforation of tympanic membrane compared with all other pathogens. None of the AOM episodes caused by M. catarrhalis was associated with mastoiditis., Conclusions: Compared with AOM caused by other pathogens, AOM caused by M. catarrhalis is characterized by a higher proportion of mixed infections, younger age at diagnosis, a lower proportion of spontaneous perforation of the tympanic membrane, and no mastoiditis.

Upper respiratory tract infections are caused by the synergistic and antagonistic interactions between upper respiratory tract viruses and 3 predominant bacterial pathogens: Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis, which are members of the commensal flora of the nasopharynx. For many bacterial pathogens, colonization of host mucosal surfaces is a first and necessary step in the infectious process. S. pneumoniae and H. influenzae have intricate interactions in the nasopharynx. The host innate immune response may influence these interactions and therefore influence the composition of the colonizing flora and the invading bacteria. S. pneumoniae, nontypeable H. influenzae, and M. catarrhalis can behave as opportunistic pathogens of the middle ear when conditions are optimal. Chronic otitis media (OM) and recurrent OM include a biofilm component. Each of the 3 predominant pathogens of OM can form a biofilm and have been shown to comprise biofilms present on middle ear mucosa specimens recovered from children with recurrent or chronic OM. Some of these characterized biofilms are of mixed bacterial etiology, suggesting that progress made on single-microbe directed strategies for treatment and/or prevention of OM, although highly encouraging, are likely to be inadequate. A significantly greater understanding about microbial physiology is required as it relates to the involvement of biofilms in OM, to identify points in the natural course of the disease that are perhaps more amenable to treatment strategies, as well as to identify biofilm-relevant antigenic targets that would be helpful in the rational design of vaccines to prevent OM.

Objectives: The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease., Methods: Biofilm formations by NTHi clinical isolates from pediatric AOM patients were evaluated by a crystal violet microtiter plate and a 98 well pin-replicator assay with a confocal laser scanning microscopy (CLSM). Optical density values of clinical isolates were compared with a positive control and the ratio of clinical isolates to a positive control was defined as biofilm formation index (BFI)., Results: 84.3% clinical isolates of NTHi were biofilm forming strains (BFI> or =0.4). The BFI represented the levels of biofilm formation and adherence on the surface. The identical strains isolated from both middle ear fluids (MEFs) and nasopharynx showed biofilm formation at the same level. The prevalence of biofilm forming isolates was significantly higher among the susceptible strains than resistant strains. The level of biofilm formation of NTHi isolated from AOM cases who was not improved by amoxicillin (AMPC) was significantly higher than that of NTHi isolated from AOM cases who was improved by AMPC., Conclusion: We clearly showed the biofilm formation of clinical NTHi isolates from AOM children. In addition, the biofilm formed by NTHi would play an important role in persistent or intractable clinical course of AOM as a result of lowered treatment efficacy of antibiotics.

Objectives: Biofilms have been implicated in the development of several chronic infections. We sought to demonstrate middle ear pathogens in adenoid biofilms using scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with confocal laser scanning microscopy (CLSM)., Methods: Comparative micro-anatomic investigation of adenoid mucosa using SEM and FISH with confocal scanning laser microscopic (CLSM) imaging from patients with recurrent acute otitis media (RAOM)., Results: All otitis-prone children demonstrated biofilm surface area presence greater than 85% by SEM. FISH accompanied by CLSM imaging also demonstrated patchy biofilms All biofilms contained middle ear pathogens and were frequent in polymicrobial distributions: 4 of 6, 4 of 6 and 3 of 6 samples contained Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, respectively., Conclusions: Dense adenoid biofilms may act as a reservoir for reinfection of the tubotympanum. Aspiration of planktonic middle ear pathogens existing in resistant adenoid biofilms during a viral upper respiratory tract infection may be an important event in the development of RAOM.

Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

Haemophilus influenzae is a human-specific gram-negative coccobacillus that causes a variety of human infections ranging from localized respiratory infections to invasive diseases. Hsf is the major nonpilus adhesin in encapsulated strains of H. influenzae and belongs to the trimeric autotransporter family of proteins. The Hsf protein contains two highly homologous binding domains, designated HsfBD1 and HsfBD2. In this study we characterized the differential binding properties of HsfBD1 and HsfBD2. In assays using HeLa cells, we found that bacteria expressing either full-length Hsf or HsfBD1 by itself adhered at high levels, while bacteria expressing HsfBD2 by itself adhered at low levels. Immunofluorescence microscopy and a cellular enzyme-linked immunosorbent assay using purified proteins revealed that the binding affinity was significantly higher for HsfBD1 than for HsfBD2. Purified HsfBD1 was able to completely block adherence by bacteria expressing either HsfBD1 or HsfBD2, while purified HsfBD2 was able to block adherence by bacteria expressing HsfBD2 but had minimal activity against bacteria expressing HsfBD1. Conversion of the residue at position 1935 in the HsfBD1 binding pocket from Asp to Glu resulted in HsfBD2-like binding properties, and conversion of the residue at position 569 in the HsfBD2 binding pocket from Glu to Asp resulted in HsfBD1-like binding properties, as assessed by adherence assays with recombinant bacteria and by immunofluorescence microscopy with purified proteins. This work demonstrates the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of the HsfBD1 and HsfBD2 binding domains.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

Nontypable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. Adherence to respiratory epithelium is an important step in the process of colonization and is influenced by adhesive proteins called adhesins. In approximately 80% of nontypable H. influenzae isolates, the major adhesins are related proteins called HMW1 and HMW2. Here, we summarize recent advances in our understanding of HMW1 and HMW2 as prototype members of the bacterial two-partner secretion pathway and examples of the expanding number of bacterial glycoproteins, highlighting experimental approaches that might be useful in studies of other secreted proteins and glycoproteins.

Objectives/hypothesis: Viral upper respiratory tract infections are often followed by secondary bacterial infections in the form of acute rhinosinusitis. We investigate the effect of rhinovirus infection on the expression of cell adhesion molecules and bacterial adherence to primary human nasal epithelial cells., Methods: Cells were infected with rhinovirus serotype 16 (RV-16), and then Staphylococcus aureus, Streptococcus pneumoniae, or Hemophilus influenzae were added to the culture. Rhinovirus-induced expression of fibronectin, platelet-activating factor receptor, and carcinoembryonic antigen-related cell adhesion molecule, was assayed by confocal microscopy, real-time polymerase chain reaction, and Western blot analysis. Bacterial adhesion to cells was assessed by confocal microscopy and the fluorescence intensity of adherent bacteria was analyzed using Image-Pro Plus 5.1 (Media Cybernetics, Inc., Bethesda, MD)., Results: RV-16 infection significantly increased the gene and protein expression of fibronectin, platelet-activating factor receptor, and carcinoembryonic antigen-related cell adhesion molecule in nasal epithelial cells. Compared with rhinovirus-uninfected control cells, the adhesion of S. aureus, S. pneumoniae, and H. influenzae increased significantly to 2.53-fold, 1.51-fold, and 2.74-fold of control levels, respectively, in rhinovirus-infected nasal epithelial cells., Conclusions: These findings suggest that increased expression of host cell adhesion molecules may be the mechanism accounting for the increase in susceptibility to bacterial rhinosinusitis associated with rhinovirus-induced upper respiratory infections.

Biofilm is a structured community of bacterial cells included in a self-produced polymeric matrix adherent to an inert or living surface. The main property of biofilm consists of making microrganisms more resistant to exogenous insults. Antibiotic therapy typically resolves symptoms determined by planktonic cells released by biofilms but is not able to eradicate and completely clear biofilm. This is why infections sustained by biofilm-producer bacteria are often recurrent, making mandatory repeated antibiotic treatments. The typical conformation of biofilm, the phenotypical and genetical features existing among the different microrganisms confer a natural resistance to a number of antimicrobials so that it is necessary to test antimicrobial activity against the microbial species itself and also against biofilm, when it is present. Comparative studies, performed on quinolones and beta-lactams, evidenced a significant activity against biofilm produced by pneumococci, haemophyli and pseudomonas as well.

The choice of empirical treatment of nosocomial pneumonia in the intensive-care unit (ICU) used to rely on the interval after the start of mechanical ventilation. Nowadays, however, the question of whether in fact there is a difference in the distribution of causative pathogens is under debate. Data from 308 ICUs from the German National Nosocomial Infection Surveillance System, including information on relevant pathogens isolated in 11,285 cases of nosocomial pneumonia from 1997 to 2004, were used for our evaluation. Each individual pneumonia case was allocated either to early- or to late-onset pneumonia, with three differentiation criteria: onset on the 4th day, the 5th day, or the 7th day in the ICU. The frequency of pathogens was evaluated according to these categories. A total of 5,066 additional cases of pneumonia were reported from 2005 to 2006, after the CDC criteria had been modified. From 1997 to 2004, the most frequent microorganisms were Staphylococcus aureus (2,718 cases, including 720 with methicillin [meticillin]-resistant S. aureus), followed by Pseudomonas aeruginosa (1,837 cases), Klebsiella pneumoniae (1,305 cases), Escherichia coli (1,137 cases), Enterobacter spp. (937 cases), streptococci (671 cases), Haemophilus influenzae (509 cases), Acinetobacter spp. (493 cases), and Stenotrophomonas maltophilia (308 cases). The order of the four most frequent pathogens (accounting for 53.7% of all pathogens) was the same in both groups and was independent of the cutoff categories applied: S. aureus was first, followed by P. aeruginosa, K. pneumoniae, and E. coli. Thus, the predictabilities of the occurrence of pathogens were similar for the earlier (1997-to-2004) and later (2005-to-2006) time frames. This classification is no longer helpful for empirical antibiotic therapy, since the pathogens are the same for both groups.

Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-gamma)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN-gamma or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.

Respiratory infections caused by nontypeable Haemophilus influenzae (NTHi) are a major medical problem. Evidence suggests that the ability to form biofilms on mucosal surfaces may play a role in NTHi pathogenesis. However, the factors that contribute to NTHi biofilm cohesion remain largely unknown. In this study we investigated the biofilm growth and detachment phenotypes of eight NTHi clinical strains in vitro. We found that the majority of strains produced biofilms within 6h when cultured statically in tubes. Biofilm formation was inhibited when culture medium was supplemented with proteinase K or DNase I. Both enzymes also caused significant detachment of pre-formed NTHi biofilms. These findings indicate that both proteinaceous adhesins and extracellular DNA contribute to NTHi biofilm cohesion. Treatment of NTHi biofilms cultured in centrifugal filter devices with DNase I, but not with proteinase K, caused a significant decrease in fluid convection through the biofilms. These results suggest that extracellular DNA is the major volumetric component of the NTHi biofilm matrix. Mechanical or enzymatic disruption of NTHi biofilms cultured in microtiter plates significantly increased their sensitivity to killing by SDS, cetylpyridinium chloride, chlorhexidine gluconate, povidone iodine and sodium hypochlorite. These findings indicate that biocide resistance in NTHi biofilms is mediated to a large part by the cohesive and protective properties of the biofilm matrix. Understanding the mechanisms of biofilm cohesion and biocide resistance in NTHi biofilms may lead to new methods for treating NTHi-associated infections.

Nontypeable Haemophilus influenzae (NTHI) bacteria are commensals in the human nasopharynx, as well as pathogens associated with a spectrum of acute and chronic infections. Two important factors that influence NTHI pathogenicity are their ability to adhere to human tissue and their ability to form biofilms. Extracellular polymeric substances (EPS) and bacterial appendages such as pili critically influence cell adhesion and intercellular cohesion during biofilm formation. Structural components in the outer cell membrane, such as lipopolysaccharides, also play a fundamental role in infection of the host organism. In spite of their importance, these pathogenic factors are not yet well characterized at the nanoscale. Here, atomic force microscopy (AFM) was used in aqueous environments to visualize structural details, including probable Hif-type pili, of live NTHI bacteria at the early stages of biofilm formation. Using single-molecule AFM-based spectroscopy, the molecular elasticities of lipooligosaccharides present on NTHI cell surfaces were analyzed and compared between two strains (PittEE and PittGG) with very different pathogenicity profiles. Furthermore, the stiffness of single cells of both strains was measured and subsequently their turgor pressure was estimated.

The adhesin protein E (PE) of the human respiratory pathogen nontypeable Haemophilus influenzae (NTHi) exists in all clinical isolates. In the present study, NTHi adherence to epithelial cells of various origins was further analyzed. The number of intraepithelial PE-deficient NTHi was decreased compared with PE-expressing NTHi. Interestingly, PE-expressing NTHi or Escherichia coli transformants, in addition to soluble recombinant PE22-160 without a lipid moiety, induced a proinflammatory cell response. The adhesive PE domain was defined within PE84-108, and preincubation of epithelial cells with this peptide blocked adhesion of several clinical NTHi isolates. Mice immunized with PE84-108 cleared NTHi up to 8-fold more efficiently on pulmonary challenge than did mice immunized with a control peptide. Finally, anti-PE mouse antibodies from vaccinated mice prevented NTHi adhesion. Our data suggest that the ubiquitous adhesin PE plays an important role in the pathogenesis of NTHi infection.

Haemophilus influenzae and Streptococcus pyogenes colonize overlapping regions of the nasopharynx and oropharynx. The effect of 10 H. influenzae strains (5 non-piliated parent strains and 5 piliated daughter strains) on adherence of S. pyogenes to human respiratory epithelial cells was assessed in this study. A 39.6% and 49.0% increase in the number of adherent S. pyogenes was observed in Chang epithelial cells if the cells were already colonized with 2 out of 5 non-piliated strains of H. influenzae (p <0.05). Four out of 5 piliated strains of H. influenzae enhanced the adherence of S. pyogenes to Chang cells by 103.5%, 113.6%, 109.7% (p<0.01) and 53.4% (p<0.05). With A549 epithelial cells, non-piliated strains of H. influenzae did not affect the adherence of S. pyogenes (p>0.05), but a 36.9% (p<0.05), 62.9% (p<0.01) and 32.1% (p<0.05) increase in the number of adherent S. pyogenes was found with 3 out of 5 piliated strains of H. influenzae. These results indicate that increases of S. pyogenes adherence to epithelial cells may be modulated species-specifically by H. influenzae, especially by piliated H. influenzae.

The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adherence to the respiratory epithelium. In this report, we show that the structure of Hia is characterized by a modular architecture containing repeats of structurally distinct domains. Comparison of the structures of HiaBD1 and HiaBD2 adhesive repeats and a nonadhesive repeat (a novel fold) shed light on the structural determinants of Hia adhesive function. Examination of the structure of an extended version of the Hia translocator domain revealed the structural transition between the C-terminal translocator domain and the N-terminal passenger domain, highlighting a highly intertwined domain that is ubiquitous among trimeric autotransporters. Overall, this study provides important insights into the mechanism of Hia adhesive activity and the overall structure of trimeric autotransporters.

Purpose: We hypothesized that observation and analysis of microbiological laboratory statistics from patients with suspected bacterial conjunctivitis should increase our understanding of microbiological epidemiology of the disease in age categories. We further assumed that the statistical data should expose evolution of antimicrobial resistance that may eventually have an influence on clinical decisions., Materials and Methods: We analyzed statistical data of bacterial isolates (1139 strains) and their resistance to common antibiotics from 2494 patients with suspected bacterial conjunctivitis., Results: Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus displayed their presence in 0- to 5-year-old children. Staphylococcus aureus and Pseudomonas aeruginosa were the most common in the elderly (>age 70) among whom a rapid increase in resistance of Staphylococcus aureus to methicillin (MRSA) was recognized., Conclusions: Our study demonstrates that the spectrum of conjunctival bacteria varies among age groups. In addition, our results confirm that a shift in antimicrobial susceptibility can be rapid and age-group specific, thus emphasizing the need for continuous surveillance of bacterial findings.

Many publications state that nontypeable Haemophilus influenzae (NTHi) produces biofilms. Here, we review many of the publications that have led to acceptance by some that NTHi expresses a biofilm-specific phenotype as a distinct part of its life cycle. Biofilm formation was originally invoked to explain the failure to culture NTHi from middle-ear effusions, recalcitrance to antibiotics and its pathogenic behaviour. We argue that the current evidence for NTHi biofilm formation in vitro and in vivo is inconclusive. We consider that NTHi biofilm is hypothesis not fact, and although it might yet prove to be correct, there has been little or no consideration of alternative interpretations for the in vitro and in vivo observations. Uncritical acceptance of a distinctive NTHi biofilm phenotype has the potential to mislead and could confuse and compromise research efforts aimed at improving management and prevention of NTHi diseases of the human respiratory tract.

Biofilms can be defined as communities of microorganisms attached to a surface. Those bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in respiratory infections, as it forms biofilms both in vitro and in vivo such as human middle ear. Recent reports indicate that otitis media, paranasal sinusitis and lower respiratory tract infections caused by Haemophilus influenzae have become more difficult to treat with oral antibiotic therapy. However, there has been no attention given to antibiotic eradication of NTHi biofilm. To investigate the antimicrobial effect of various antibiotics against NTHi biofilm formation, we conducted the following comparative study using both beta-lactamase-negative ampicillin (AMP)-susceptible (BLNAS) and AMP-resistant (BLNAR) NTHi strains. In a microtiter biofilm assay, both levofloxacin and gatifloxacin, of the fluoroquinolone antibiotic group, significantly inhibited biofilm formation by BLNAS and BLNAR NTHi in a dose-dependent fashion compared to ampicillin of the penicillin antibiotic group, cefotaxime of the cephalosporin antibiotic group, and both erythromycin and clarithromycin of the macrolide antibiotic group. Furthermore, in flow cell chamber studies, confocal laser scanning microscopy counted survival bacteria in mature biofilm had been treated with gatifloxacin, ampicillin, cefotaxime and erythromycin. Only gatifloxacin completely killed the BLNAR NTHi isolates within biofilms without regard to the thickness of biofilm formation. The results of this study suggest that fluoroquinolones potentially have a role in therapy against diseases caused by both BLNAS and BLNAR NTHi isolates within biofilms.

Non-typable Haemophilus influenzae (NTHi) is an important human-specific respiratory pathogen colonizing the mucosa of the upper respiratory tract. The bacterium is a common cause of acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease (COPD). An immunoglobulin (Ig) D-lambda myeloma protein was found to detect a 16 kDa surface protein that we designated protein E (PE). The pe gene was cloned using an NTHi genomic DNA library, and a truncated PE-derived protein lacking the endogenous signal peptide (PE22-160) was synthesized and produced in large amounts in Escherichia coli. Interestingly, PE was expressed at the bacterial surface of NTHi as revealed by flow cytometry using the IgD-lambda myeloma protein or PE-specific polyclonal antibodies. A PE-deficient NTHi mutant was produced and lost 50% of its adhesive capacity as compared to the wild-type counterpart when analysed for adhesion to type II lung alveolar epithelial cells. In parallel, E. coli expressing full-length PE1-160 adhered significantly more efficiently to epithelial cells as compared to wild-type E. coli. Recombinant IgD that recognized the chemical dansyl-chloride did not interact with PE indicating that the IgD-lambda myeloma protein most likely was an antibody directed against the H. influenzae surface epitope. In conclusion, we have discovered a novel NTHi outer membrane protein with adhesive properties using an IgD-myeloma protein.

In vitro studies suggest an important role for CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) in infection by multiple gram-negative bacteria. However, in vivo evidence supporting this role is lacking, largely because the bacterial adhesins involved in this host-microbe association do not bind to murine-derived CEACAM1. One of several adhesins expressed by nontypeable Haemophilus influenzae (NTHI), the outer membrane protein P5-homologous adhesin (or P5), is essential for colonization of the chinchilla nasopharynx and infection of the middle ear. Here we reveal that NTHI P5 binds to the chinchilla homologue of CEACAM1 and that rabbit anti-human carcinoembryonic antigen blocks NTHI colonization of the chinchilla nasopharynx, providing the first demonstration of a role for CEACAM receptor binding by any bacterial pathogen in vivo.

Objective: The objective of the present study was to identify bacterial biofilms in tissue samples obtained from paediatric patients undergoing surgical treatment, for recurrent upper airway infections, frequently associated to effusive otitis media, unresponsive to repeated cycles of selective medical antibiotic and anti-inflammatory therapy and assay the ability of Haemophilus influenzae strains, most frequently identified in our cultural examinations, to grow as biofilm in vitro., Methods: We examined 18 surgical specimens (18 adenoids) from the upper respiratory tract, obtained from 18 paediatric patients. Tissues were cultured using conventional methods and subjected to scanning electron microscopy for detection of biofilm. Haemophilus influenzae strains, were cultured on 96-sterile well polystyrene microtiter plates (CELLSTAR-greiner bio-one) and stained with 1% crystal violet to quantify biofilm production., Results: Bacterial cocci attached to the tissue surface and organized in colonies, with a morphology consistent with bacterial coccoid biofilms, were observed in all adenoid (18/18) samples. Haemophilus influenzae isolates from 11/18 (61.1%) of our tissue samples scored a percentual transmittance (%Tbloc) > 50, identifying a high capacity to form biofilms (level 4)., Conclusions: Bacterial biofilms identified in adenoid tissue of paediatric patients with recurrent upper airway inflammatory processes, associated to chronic effusive otitis media, may represent a bacterial "reservoir" responsible of the maintenance of chronic inflammatory mucosal reactions, resistant to selective antibiotic therapy and requiring surgical treatment.

The recently reported structures of the bacterial multidrug exporter Sav1866 suggest a domain architecture in which both nucleotide-binding domains (NBDs) of this ATP binding cassette (ABC) transporter contact both transmembrane domains (TMDs). Such a domain arrangement is particularly unexpected because it is not found in the structures of three solute importers BtuCD, HI1470/1, and ModBC from the same protein family. There is also no precedent for such an arrangement from biochemical studies with any ABC transporter. Sav1866 is homologous with the clinically relevant human P-glycoprotein (ABCB1). If the structure proposed for Sav1866 is physiologically relevant, the long intracellular loops of P-glycoprotein TMD2 should contact NBD1. We have tested this by using cysteine mutagenesis and chemical cross-linking to verify proximal relationships of the introduced sulfhydryls across the proposed interdomain interface. We report the first biochemical evidence in support of the domain arrangement proposed for the multidrug resistance class of ABC transporters. With a domain arrangement distinctly different from the three solute importers it seems likely that the TMDs of ABC importers and exporters have evolved different mechanisms to couple to common conformational changes at conserved NBDs.

The physiological roles of the glutathione(GSH)/glutathione peroxidase(GPx) system in protecting microbial cells against oxidative stress were reviewed. In eukaryotic model microbe Saccharomyces cerevisiae,this system is obligatory in maintaining the redox balance and defending the oxidative stress. However, the GSH/GPx system only conditionally exists in prokaryotes. Namely,for those prokaryote bacteria containing glutathione reductase and GPx, e.g. Haemophilus influenzae and Lactococcus lactis, by taking up GSH, they might develop a conditional GSH-dependent GPx reduction system, which conferred cells a stronger resistance against oxidative challenge.

Respiratory syncytial virus (RSV) infection is associated with secondary bacterial infections caused by nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. The pathogenesis of these complications is not completely understood; however, viral infection of respiratory epithelial cells promotes colonization by these bacteria. In the present study, RSV virions associated with NTHi and pneumococci in an inoculum-dependent manner in a fluid-phase binding assay. Adherence of NTHi and S. pneumoniae to epithelial cells transiently expressing RSV G glycoprotein was 2- and 2.2-fold higher, respectively, than adhesion to cells transfected with the vector alone (P <0.01). Furthermore, 4.6- and 6.2-fold larger numbers of NTHi and pneumococci bound to cells expressing a membrane-bound full-length RSV G protein than to cells expressing a truncated non-membrane-bound protein (P

Mucin, a major component of mucus, plays a critical role in host mucosal defense response by participating in mucociliary clearance. However, if overproduced, overproduced mucus leads to airway mucus obstruction and conductive hearing loss. Despite extensive studies that focus on investigating how MUC5AC mucin is regulated by one inducer at a time, how MUC5AC is synergistically regulated by multiple factors remains unknown. Here we provide direct evidence for the first time that bacterial pathogen NTHi and human growth factor EGF synergize with each other to potently up-regulate MUC5AC mucin transcription. Moreover, activation of both p38 and ERK is required for synergistic induction of MUC5AC by NTHi and EGF. Finally, PAK2 and PAK4 are differentially involved in this synergistic induction of MUC5AC by acting upstream of p38 and ERK. Our studies bring novel insights into our understanding of synergistic regulation of MUC5AC mucin by both pathological and physiological inducers.

The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.

The non-typeable Haemophilus influenzae strain DH1 was isolated from a 25 year old male patient with Fisher syndrome, a postinfectious autoimmune condition characterized by the presence of anti-GQ1b IgG antibodies that target and initiate damage to peripheral nerves. DH1 was found to display an alphaNeuAc(2-8)alphaNeuAc(2-3)betaGal branch bound to the tetraheptosyl backbone core of its lipooligosaccharide (LOS). The novel sialylation pattern was found to be dependent on the activity of a bifunctional sialyltransferase, Lic3B, which catalyzes the addition of both the terminal and subterminal sialic acid residues. Patient serum IgGs bind to DH1 LOS, and the reactivity is significantly influenced by the presence of sialylated glycoforms. The display by DH1, of a surface glycan that mimics the terminal trisaccharide portion of disialosyl-containing gangliosides, provides strong evidence for its involvement in the development of Fisher syndrome.

The incidence of mixed viral/bacterial infections has increased recently because of the dramatic increase in antibiotic-resistant strains, the emergence of new pathogens, and the resurgence of old ones. Despite the relatively well-known role of viruses in enhancing bacterial infections, the impact of bacterial infections on viral infections remains unknown. In this study, we provide direct evidence that nontypeable Haemophilus influenzae (NTHi), a major respiratory bacterial pathogen, augments the host antiviral response by up-regulating epithelial Toll-like receptor 7 (TLR7) expression in vitro and in vivo. Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAK-TRAF6-IKK-NF-kappaB-dependent signaling pathway. Interestingly, CYLD, a novel deubiquitinase, acts as a negative regulator of TLR7 induction by NTHi. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response and further identifies CYLD for the first time as a critical negative regulator of host antiviral response.

Aim of the present study was to identify bacterial biofilms in tissue samples obtained from paediatric patients undergoing surgical treatment, for chronic and recurrent adeno-tonsillitis, not responding to repeated cycles of selective medical antibiotic and anti-inflammatory treatment and to assay the ability of Haemophilus influenzae strains, most frequently identified in the culture examinations, to grow as biofilm in vitro. Overall, 25 surgical specimens (15 adenoids, 10 tonsils) were examined from the upper respiratory tract, from 15 paediatric patients (mean age 6 years). All patients were affected by recurrent and/or chronic adenoiditis and adenotonsillitis unresponsive to selective antibiotic and anti-inflammatory therapy. Tissues were cultured using conventional methods and subjected to scanning electron microscopy for detection of biofilm. Haemophilus influenzae strains, were cultured on 96-sterile well polystyrene microtitre plates (CELLSTAR-greiner bio-one) and stained with 1% crystal violet to quantify biofilm production. Bacterial cocci attached to the tissue surface and organized in colonies, with a morphology consistent with bacterial coccoid biofilms, were observed in all adenoid (15/15) and in 6/10 tonsil samples. Haemophilus influenzae isolates from 12/25 (48%) of our tissue samples scored a percent transmittance (%T(bloc)) > 50, displaying a high capacity to form biofilms (level 4). In conclusion identification of bacterial biofilms in chronic and/or recurrent paediatric upper airway inflammatory processes and the capacity to produce biofilm in vitro, demonstrated by Haemophilus influenzae (the most frequently identified bacteria in our samples), could be related to the aetiopathogenic role of biofilms in chronic inflammatory mucosal reactions and to the resistance of these infections to selective antibiotic therapy.

Purpose: Antigen-sampling M cells have been identified in conjunctival tissue overlying lymphoid follicles in rabbits and guinea pigs. Conjunctival M cells in the guinea pig display alpha(2-3) sialic acid on their surfaces, as evinced by selective labeling by Maackia amurensis leukoagglutinin (MAL)-I. Haemophilus influenzae strains OM12, which expresses the HMW1 adhesin for alpha(2-3) sialic acid, and Rd KW20, which lacks HMW1, were used to test the hypothesis that conjunctival M cells translocate large microbes., Methods: Fluorescein-labeled bacteria were instilled into the conjunctival sac for up to 130 minutes. Confocal laser scanning microscopy and electron microscopy were used to visualize bacterial distribution., Results: M cells, but not nonfollicular epithelial cells in the palpebral region, selectively bound and translocated bacteria. By 66 minutes, 423 +/- 165 bacteria/mm(2) of follicle-associated epithelial (FAE) surface were found in three-dimensional reconstructions extending 15.4 mum below the surface. By 127 minutes, the number of bacteria increased to 579 +/- 44/mm(2) of FAE surface and they had moved 50% deeper into the follicle. Coadministration with MAL-I reduced OM12 transport by 61%. Similarly, Rd KW20 uptake was 71% less at 63 minutes and 58% less at 121 minutes, indicating that OM12 uptake is at least partially mediated by binding to alpha(2-3) sialic acid., Conclusions: Conjunctival M cells are a port of entry for large microbes and may play a role in initiation of mucosal immune responses against commensal or transient ocular bacterial species and may allow the entry of pathogens.

In the present study, we followed the CLSI procedure M40-A to evaluate three specimen transport systems [the new BD CultureSwab MaxV(+), the new Remel BactiSwab, and the Medical Wire & Equipment Transwab] for the survival of fastidious and nonfastidious organisms for 0, 6, 24, and 48 h at room temperature. BD CultureSwab MaxV(+) outperformed the other two swabs for the recovery of the three fastidious organisms, Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis for up to 48 h. Indeed, BD CultureSwab MaxV(+) maintained a constant number of viable H. influenzae and N. meningitidis for up to 48 h, and only a 2 log reduction was noted for N. gonorrhoeae, fulfilling the requirements of M40-A guidelines. However, unlike Remel BactiSwab and the Medical Wire & Equipment Transwab, which fulfilled the M40-A requirements for maintaining the viability of Streptococcus pneumoniae, BD CultureSwab MaxV(+) could not maintain the viability of S. pneumoniae reference or clinical strains past 6 h. Excellent overall sensitivity (98%) (95% confidence interval, 89.5 to 99.7) was observed when the BD CultureSwab MaxV(+) rectal swabs were compared to the "gold standard" stool cultures. Thus, the BD CultureSwab MaxV(+) rectal swab can be used when investigating gastrointestinal bacterial outbreaks or when health care providers face difficulties in obtaining stool samples, particularly from children.

We have previously shown that C3 binding to serum-resistant nontypeable Haemophilus influenzae (NTHi) strain R2866 is slower than C3 binding to a serum-sensitive strain. Ab-dependent classical pathway activation is required for complement-dependent killing of NTHi. To further characterize the mechanism(s) of serum resistance of R2866, we compared binding of complement component C4b to R2866 with a serum-sensitive variant, R3392. We show that C4b binding to R2866 relative to R3392 was delayed, suggesting regulation of the classical pathway of complement. Increased C4b deposition on R3392 was independent of the amount and subclass of Ab binding, suggesting that an impediment to C4b binding existed on R2866. Immunoblotting and mass spectrometry indicated that lipooligosaccharide and outer membrane proteins P2 and P5 were targets for C4b. P2 and P5 sequences and expression levels were similar in both strains. Insertional inactivation of the phase-variable lipooligosaccharide biosynthesis gene lgtC in R2866 augmented C4b deposition to levels seen with R3392 and rendered the bacteria sensitive to serum and whole blood. These results suggest a direct role of lgtC expression in the inhibition of C4b deposition and consequent serum resistance of R2866. Alteration of surface glycans of NTHi may be a critical event in determining the ability of a strain to evade host defenses and cause disseminated infection.

Haemophilus influenzae is a cause of otitis media with effusion (OME). Animal models demonstrate growth of H. influenzae biofilms in OME, which may explain why OME does not respond well to conventional antibiotic therapy. Using a previously developed in vitro model, we performed H. influenzae susceptibility studies to see if H. influenzae biofilm cultures were more resistant to antibiotics than planktonic (broth) cultures, and to determine which antibiotics were most effective against H. influenzae biofilms. H. influenzae isolates were grown as biofilms on polystyrene pins. Biofilm and planktonic minimum inhibitory concentrations (MICs) were measured for 8 antibiotics, and multiple combination testing was performed with 66 groupings of 1, 2, or 3 antibiotics. We found that biofilm cultures were more resistant to antibiotics than planktonic ones. Antibiotic combinations containing rifampin and ciprofloxacin were most effective against biofilms. Biofilm testing reveals differences in effectiveness among antibiotics not apparent from conventional susceptibility testing, and suggests novel antibiotic regimens that could be studied for treatment of OME.

Rhinovirus (RV) is an important trigger of chronic obstructive pulmonary disease (COPD) exacerbations. In addition, respiratory viruses are more likely to be isolated in patients with a history of frequent exacerbations, suggesting that these patients are more susceptible to viral infection. To examine potential mechanisms for cooperative effects between bacterial and viral infection in COPD, we studied the responses of cultured human airway epithelial cells to nontypeable Hemophilus influenzae and RV. In both 16HBE14o- and primary mucociliary-differentiated cells, preincubation with H. influenzae enhanced RV serotype 39-induced protein expression of interleukin (IL)-8, epithelial-derived neutrophil attractant-78, and growth-related oncogene-alpha. H. influenzae infection also increased the binding of RV39 to cultured cells, as well as expression of intercellular adhesion molecule (ICAM)-1 and Toll-like receptor (TLR)-3, receptors for RV and dsRNA, respectively. Neutralizing antibody against tumor necrosis factor-alpha inhibited IL-8 expression induced by H. influenzae and RV39. Finally, siRNA against TLR3 attenuated RV-induced IL-8 expression. We conclude that H. influenzae infection increases airway epithelial cell ICAM-1 and TLR3 expression, leading to enhanced binding of RV and a potentiation of RV-induced chemokine release. These data provide a cellular mechanism by which H. influenzae infection may increase the susceptibility of COPD patients to RV-induced exacerbations.

With the elimination of Haemophilus influenzae type b through vaccination, it has been suggested that other types of H. influenzae strains might acquire virulence traits and emerge as important pathogens. The gene sequence IS1016 has been associated with an increased capacity to cause severe infections. It is usually present in encapsulated strains but is sometimes harbored by nontypeable H. influenzae strains. To explore this further, 118 H. influenzae isolates, collected from both patients and healthy carriers, were investigated with PCR with reference to this gene sequence. Isolates positive for the insertion element were bio- and serotyped. The presence of hmw genes for adherence, the genetic profile, and the ability to form biofilm in vitro were investigated. A total of 15 isolates were IS1016-positive, whereof 12 were nontypeable. All 12 nontypeable isolates were obtained from healthy carriers, and 92% of the isolates were biotype I. They cross-reacted to some extent with type-specific antisera or exhibited a restricted genetic diversity like encapsulated strains. Furthermore, they lacked hmw-genes, and their ability to form biofilms was comparable with a capsule-deficient type b strain. Although this subset of strains mimicked traits usually exhibited by encapsulated strains, the isolation frequency did not seem to have been affected by vaccination.

In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimeric-autotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity.

Rationale: Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF), especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF., Objectives: This study investigated ability of NTHi to form biofilms and its impact on airway epithelia using in vivo and in vitro analyses., Methods: We evaluated bronchoalveolar lavage fluid from young patients with CF for evidence of NTHi biofilms. To further investigate the pathogenesis of NTHi in respiratory infections, we developed a novel in vitro coculture model of NTHi biofilm formation on polarized human airway epithelial cells grown at the air-liquid interface., Measurements and Main Results: In bronchoalveolar lavage fluid samples from young, asymptomatic patients with CF, we found morphologic evidence suggestive of NTHi biofilm formation. In addition, 10 clinical NTHi isolates from patients with CF formed biofilms on plastic surfaces. NTHi formed biofilms on the apical surface of cultured airway epithelia. These biofilms exhibited decreased susceptibility to antibiotics and were adherent to epithelial surfaces. Airway epithelial cells remained viable throughout 4 d of coculture, and responded to NTHi with nuclear factor-kappaB signaling, and increased chemokine and cytokine secretion., Conclusions: NTHi formed adherent biofilms on the apical surface airway epithelia with decreased susceptibility to antibiotics, and respiratory cells exhibited inflammatory and host defense responses-evidence of a dynamic host-pathogen interaction. The data presented here have implications both for understanding early CF lung disease pathogenesis and for the treatment of early, asymptomatic colonization of patients with CF with H. influenzae.

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.

The ability to preserve the fragile ultrastructural organization of bacterial biofilms using cryo-preparation methods for electron microscopy has enabled us to probe sections through non-typeable Haemophilus influenzae (NTHi) biofilms and determine the localization of NTHi-specific lipooligosaccharide (LOS) and proteins within these structures. Some of the proteins we examined are currently being considered as candidates for vaccine development, so it is important that their distribution and accessibility within the biofilms formed by NTHi be determined. We have localized LOS to the extracellular matrix (ECM) of the biofilm and the P6 outer membrane protein to the membrane of what appear to be viable bacteria within the biofilm. The Hap and HWM1/HMW2 adhesive proteins were associated with bacteria within the biofilm and were present in the biofilm ECM. The IgA1 protease is a secreted protein that was also associated with NTHi in the biofilm and was in the ECM, but was more concentrated in the top region of the biofilm, suggesting a role in protecting biofilm bacteria from antibody attack.

The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, we characterized the functional domains of the Haemophilus influenzae HMW1B protein, a TpsB protein that interacts with the H. influenzae HMW1 adhesin. Using c-Myc epitope tag insertions and cysteine substitution mutagenesis, we discovered that HMW1B contains an N-terminal surface-localized domain, an internal periplasmic domain, and a C-terminal membrane anchor. Functional and biochemical analysis of the c-Myc epitope tag insertions and a series of HMW1B deletion constructs demonstrated that the periplasmic domain is required for secretion of HMW1 and that the C-terminal membrane anchor (HMW1B-(234-545)) is capable of oligomerization and pore formation. Similar to our observations with HMW1B, examination of a Bordetella pertussis TpsB protein called FhaC revealed that the C terminus of FhaC (FhaC-(232-585)) is capable of pore formation. We speculate that all TpsB proteins have a modular structure, with a periplasmic domain that interacts with the cognate TpsA protein and with pore forming activity contained within the C terminus.

Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure-function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.

Nontypeable Haemophilus influenzae (NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation, PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated with PCho substitutions. We thus conclude that biofilm growth increases the proportion of PCho+ variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.