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Otitis media with effusion (OME) is a common inflammatory disease that primarily affects children. OME is defined as a chronic low-grade inflammation of the middle ear (ME), without any signs of infection and with effusion persisting in the ME for more than 3 months. The precise pathogenesis is, however, not fully understood. Here, we comprehensively characterized and compared the host immune responses (inflammatory cells and mediators) and the overall microbial community composition (microbiota) present in matched middle ear effusion (MEE) samples, external ear canal (EEC) lavages, and nasopharynx (NPH) samples from children with OME. Female patients had significantly increased percentages of T lymphocytes and higher levels of a wide array of inflammatory mediators in their MEE compared to that of male patients, which were unrelated to microbiota composition. The relative abundances of identified microorganisms were strongly associated with their niche of origin. Furthermore, specific inflammatory mediators were highly correlated with certain bacterial species. Interestingly, some organisms displayed a niche-driven inflammation pattern in which presence of Haemophilus spp. and Corynebacterium propinquum in MEE was accompanied by proinflammatory mediators, whereas their presence in NPH was accompanied by anti-inflammatory mediators. For Turicella and Alloiococcus , we found exactly the opposite results, i.e., an anti-inflammatory profile when present in MEE, whereas their presence in the the NPH was accompanied by a proinflammatory profile. Together, our results indicate that immune responses in children with OME are highly niche- and microbiota-driven, but gender-based differences were also observed, providing novel insight into potential pathogenic mechanisms behind OME., (Copyright © 2020 Enoksson et al.)

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The pathology of AOM increases during long-term infection in the middle ear (ME), but the host cellular immune response to bacterial infection in this inflamed environment is poorly understood. Using the Junbo mouse, a characterized NTHi infection model, we analyzed the cellular response to NTHi infection in the Junbo mouse middle ear fluid (MEF). NTHi infection increased the total cell number and significantly decreased the proportion of live cells in the MEF at day 1, and this further decreased gradually on each day up to day 7. Flow cytometry analysis showed that neutrophils were the dominant immune cell population in the MEF and that NTHi infection significantly increased their proportion whereas it decreased the monocyte, macrophage, and dendritic cell proportions. Neutrophil and macrophage numbers increased in blood and spleen after NTHi infection. The T-cell population was dominated by T-helper (Th) cells in noninoculated MEF, and the effector Th (CD44 + ) cell population increased at day 2 of NTHi infection with an increase in IL-12p40 levels. Sustained NTHi infection up to 3 days increased the transforming growth factor β levels, decreasing the effector cell population and increasing the T-regulatory (T-reg) cell population. In the preinflamed ME environment of the Junbo mouse, neutrophils are the first responder to NTHi infection followed by T-reg immune suppressive cells. These data indicate that sustained NTHi infection in the ME induces the immune suppressive response by inducing the T-reg cell population and reducing immune cell infiltration, thus promoting longer-term infection., (© Crown copyright 2019.)

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3 -/- mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3 -/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3 -/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3 -/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease., (Copyright © 2017 American Society for Microbiology.)

Acute otitis media (AOM) is one of the most common bacterial infectious diseases in children aged 2 to 7 years worldwide. We previously demonstrated that interleukin-17A (IL-17A) promotes an acute inflammatory response characterized by the influx of neutrophils into the middle ear cavity during Streptococcus pneumoniae -induced AOM. In general, the inflammatory response is viewed as an effector that frequently causes local tissue damage. However, little is known about the pathogenic effects of IL-17A in AOM. Here, we investigated the pathogenic effects of IL-17A by using wild-type (WT) and IL-17A knockout (KO) mouse models. The results showed that the pathogenic effects of AOM, including weight loss, histopathological changes, and proinflammatory cytokine production, were more severe in WT mice than in IL-17A KO mice, suggesting that IL-17A aggravates tissue damage in AOM. Furthermore, these pathogenic effects were found to be dependent on p38 mitogen-activated protein kinase (MAPK) and could be reversed in the presence of a p38 MAPK-specific inhibitor. It was also demonstrated that IL-17A promoted the production of neutrophil myeloperoxidase (MPO) through the p38 MAPK signaling pathway, which was responsible for the middle ear tissue injury. These data support the conclusion that IL-17A contributes to middle ear injury through the p38 MAPK signaling pathway., (Copyright © 2017 American Society for Microbiology.)

Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapA-deficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.

To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2(-/)(-)), C1qa (C1qa(-/)(-)), and factor B (Bf(-)(/)(-)). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2(-/)(-) mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2(-/)(-) mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa(-)(/)(-) and Bf(-)(/)(-) mice, and least for Bf/C2(-)(/)(-) mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.

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.

Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho(+) variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho(+) variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.

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.

Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

Streptococcus pneumoniae strains are the leading cause of bacterial otitis media, yet little is known about specific bacterial factors important for this disease. We utilized a molecular epidemiological approach involving genomic subtraction of the S. pneumoniae serogroup 19 middle ear strain 5093 against the laboratory strain R6. Resulting subtraction PCR (sPCR) products were used to screen a panel of 93 middle ear, 90 blood, 35 carriage, and 58 cerebrospinal fluid isolates from young children to identify genes found more frequently among middle ear isolates. Probe P41, similar to a hypothetical protein of Brucella melitensis, occurred among 41% of middle ear isolates and was found 2.8 (95% confidence interval [CI], 1.32 to 6.5), 3.3 (95% CI, 1.9 to 5.7), and 1.8 (95% CI, 1.1 to 3.0) times more frequently among middle ear strains than carriage, blood, or meningitis strains, respectively. sPCR fragment H10, similar to an unknown Streptococcus agalactiae protein, was present in 31% of middle ear isolates and occurred 3.6 (95% CI, 1.2 to 11.2), 2.8 (95% CI, 1.5 to 5.4), and 2.6 (95% CI, 1.2 to 5.5) times more often among middle ear isolates than carriage, blood, or meningitis strains, respectively. These studies have identified two genes of potential importance in otitis media virulence. Further studies are warranted to outline the precise role of these genes in otitis media pathogenesis.

Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective.

Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

Previous studies have demonstrated that the Moraxella catarrhalis surface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence of M. catarrhalis to these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8- to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that the M. catarrhalis Hag protein is an adhesin for cell lines derived from human lung and middle ear tissues.

The rat middle ear and lung clearance model has been used to show that the nontypeable Haemophilus influenzae 26-kDa outer membrane protein OMP26 is highly efficacious as a mucosal immunogen, inducing significantly enhanced clearance in immunized rats upon direct challenge of these two anatomic sites. Similarly, the chinchilla model of middle ear and nasopharyngeal clearance has been used to show that two P5 fimbrin adhesin-derived immunogens, LB1 and lipoprotein D (LPD)-LB1(f)(2,1,3), are highly efficacious as parenteral immunogens. Both induced significantly augmented clearance of nontypeable H. influenzae upon challenge of these sites. Here, these three nontypeable H. influenzae immunogens in addition to six bovine serum albumin and keyhole limpet hemocyanin conjugates of the synthetic peptide LB1(f) were assayed for relative efficacy in the reciprocal rodent model system. OMP26 was assayed in the chinchilla host by a parenteral immunization route, with clearance of the middle ear and nasopharynx used as outcome measures. Both LB1 and LPD-LB1(f)(2,1,3) were assayed in the rat host with a mucosal immunization route and clearance of nontypeable H. influenzae from the lungs and middle ears as outcome measures. Both of the immunogens were found to induce a high-titered and specific immune responses in the heterologous host system. Moreover, each was found to be highly efficacious in the reciprocal host system, providing strong support for the continued development and inclusion of both OMP26 and P5 fimbrin-derived peptides as candidate vaccine antigens directed at otitis media caused by nontypeable H. influenzae.

Experimental and clinical studies suggest that influenza A virus promotes Streptococcus pneumoniae-induced otitis media; however, the mechanism underlying this synergistic interaction has not been completely defined. In this study, glycoconjugate expression patterns were evaluated on the cell surface in the chinchilla eustachian tube (ET) lumen of a cohort challenged intranasally (i.n.) with S. pneumoniae type 6A, which is predominantly transparent and a cohort with an antecedent influenza A virus infection, followed by i.n. inoculation with S. pneumoniae. The labeling patterns obtained with six lectin probes revealed that the binding of Bandeiraea simplicifolia lectin II, succinylated wheat germ agglutinin, and peanut agglutinin were significantly increased in the lumenal surface of the ET in the cohort infected with both pathogens compared to the cohort inoculated with only S. pneumoniae, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. A significant decreased labeling with Sambucus nigra agglutinin in the combined influenza A virus and pneumococcus infection cohort suggested that there were few sialic acid residues remaining in the ET epithelium. In addition, the colonial opacity of S. pneumoniae during the disease course was examined. The opaque phenotype was predominant among the pneumococcus isolates from the middle-ear fluid in the cohort infected with the both pathogens. Together, these data suggest that the synergic effect of influenza A virus and S. pneumoniae on the changes of the carbohydrate moieties in the ET epithelium and that the selection of the opaque variant may facilitate the pneumococcal invasion of the middle ear.

To define the role of nontypeable Haemophilus influenzae (NTHI) lipooligosaccharide (LOS) in the induction of proinflammatory cytokine gene expression during otitis media, we compared the abilities of formalin-killed NTHI strain 2019 and its LOS htrB and rfaD mutants to stimulate human middle ear epithelial (HMEE) cell cytokine and chemokine gene expression and production in vitro. Strain DK-1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. HMEE cells were incubated with formalin-killed NTHI 2019, B29, or DK-1. The supernatants and the cells were collected at 2, 4, 8, and 24 h after stimulation. Expression of genes for the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin lbeta (IL-1beta), and IL-6 and for the chemokines macrophage inflammatory protein 1beta (MIP-1beta), monocyte chemotactic peptide 1 (MCP-1), and IL-8 was quantitated by real-time PCR. NTHI B29 did not significantly stimulate any cytokine or chemokine mRNA expression in HMEE cells. In striking contrast, NTHI 2019 induced up to 105-, 139-, and 187-fold increases in HMEE cell expression of IL-1beta, TNF-alpha, and MIP-1beta, respectively (P < 0.01 [2019 versus B29]). NTHI 2019 also induced upregulation of IL-8, IL-6, and MCP-1 mRNA expression (by 26-, 44-, and 14-fold, respectively [P < 0.05 (2019 versus B29)]). The significant induction of cytokine genes was confirmed by quantitating the secretion of cytokines in culture supernatants with an enzyme-linked immunosorbent assay. There were no significant differences in mRNA expression of IL-8, IL-6, and MCP-1 between the 2019- and DK-1-treated groups. The low levels of gene transcripts observed after incubation of HMEE cells with B29 indicate that products of the disrupted NTHI htrB LOS gene may play a major role in induction of these particular inflammatory mediators.

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (10(7) CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-alpha) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-alpha in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-alpha production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable Haemophilus influenzae. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable H. influenzae from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five hif genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable H. influenzae associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.

The indigenous microbial flora of the middle ear cavity of Mongolian gerbils, Meriones unguiculatus, was isolated, characterized, and identified, showing it to be sparse and transitory. Organisms, when found in the middle ear cavity, were most likely to be Staphylococcus epidermidis-like organisms. Cerumen from the external auditory canal of these animals yielded mostly staphylococci, coryneforms, and other gram-positive rods, including some anaerobic species. The nasopharynx supported a flora consisting mainly of staphylococci, lactobacilli, and coryneforms, with a smaller incidence and numbers of many other species. No mycoplasmas were cultured or seen in scanning electron microscope studies. None of the major pathogens of human otitis media were found; therefore, Mongolian gerbils are microbiologically acceptable candidates as a model for induced otitis media, using organisms isolated from human otitic infections.

A semiquantitative nasopharyngeal culture was found to be sensitive and specific in predicting middle ear pathogens in children with acute bacterial otitis media. In nasopharyngeal specimens with growth of at least 1,000 colonies, the tympanocentesis isolate was present and was either the predominant isolate or accounted for 50% of growth in 16 of 16 children. Data suggest that virulence of nasopharyngeal organisms plays a role in the pathogenesis of acute otitis media. Qualitative differences were found in the nasopharyngeal flora of children with bacterial otitis media as compared with children with clinical otitis media and sterile tympanocentesis cultures, children with uncomplicated upper respiratory illness, and healthy children. Abundant growth of Haemophilus influenzae (greater than or equal to 50% total colony count) was associated with children with bacterial otitis media, and abundant Branhamella catarrhalis was associated with the others (P less than or equal to 0.01). Abundant growth of Streptococcus pneumoniae occurred frequently and regardless of clinical category. Antibiotic treatment of children with otitis media resulted in rapid quantitative and qualitative changes in nasopharyngeal flora.

Studies were performed to determine the optimal sites for primary isolation of Mycoplasma pulmonis from the respiratory system of laboratory rats. In a preliminary study, a group of 42 rats was cultured for respiratory M. pulmonis at several sites. Tracheal washes yielded the highest number of positive cultures (61.9%) and ground lung tissue yielded the lowest (19.0%), with the nasopharyngeal culture in the middle (52.38%). In a subsequent study, the tracheal wash was again the optimal method since 76.7% of 103 rats were positive for M. pulmonis, whereas culture of the nasopharyngeal area resulted in 53.9% positive cultures. In a third study, all 45 rats were positive for M. pulmonis when the tracheal wash method was used, whereas only 51% were positive when the nasopharyngeal area was cultured, indicating that the tracheal wash was the most reliable of the commonly used procedures. Other aspects of these experiments demonstrated the tympanic cavity of animals with otitis media to be an excellent source for obtaining material to culture for M. pulmonis.

Bacteria were isolated from 29 Mongolian gerbils, Meriones unguiculatus, with spontaneous aural cholesteatomas. We identified 148 cultures, 44 from the middle ear surface of cholesteatomas and 104 from cholesteatoma contents. We could only identify 63 cultures to the genus level, although we identified 85 cultures as belonging to 21 different species. We found on the surfaces of cholesteatomas representatives of 9 genera, from which 8 species could be identified, and representatives of 19 genera within the cholesteatoma sac, from which 21 species could be identified. The most common bacterial genera isolated were Staphylococcus, Pseudomonas, and Corynebacterium among the aerobic genera, and Bacteroides and Peptococcus among the anaerobic genera. The bacterial flora of gerbilline cholesteatomas was found to be diverse, resembling the flora found to be associated with human cholesteatomas. The flora also resembled the organisms found transitorily within the normal middle ear cavity of gerbils, except for a higher incidence of pseudomonads.

Seventy percent of pneumococci isolated from the middle-ear cavity of infants and children with acute otitis media were of one of the seven serotypes 1, 3, 6, 14, 18, 19, or 23. The immunological response in the serum and middle-ear fluid from otitis media caused by one of these serotypes was studied in 61 children by using either indirect hemagglutination or indirect fluorescent antibody tests, or both. Twenty-six of the patients had pneumococcal antibody present in the acute serum and 28 had it in the convalescent serum by at least one method. Thirteen of the 49 middle-ear fluids examined had antibody by the indirect fluorescent antibody technique. Serum pneumococcal antibody was found to reside predominantly in the immunoglobulin G or immunoglobulin M classes, whereas pneumococcal antibody with middle-ear fluid was found to be distributed equally among all three classes. Approximately 25% of the patients (16 of 61) had a positive immune response to their infection as evidenced by increased levels of pneumococcal antibody in the convalescent serum. The percentage of patients responding immunologically increased with age: 12% of infants less than 12 months showed a significant response, whereas 48% of children over 24 months responded.